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Zbigniew Darzynkiewicz - One of the best experts on this subject based on the ideXlab platform.
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atm activation and h2ax phosphorylation induced by genotoxic agents assessed by flow and Laser Scanning Cytometry
Methods of Molecular Biology, 2017Co-Authors: Hong Zhao, Dorota H Halicka, Jorge Garcia, Zbigniew DarzynkiewiczAbstract:Activation of Ataxia Telangiectasia Mediated protein kinase (ATM) by its phosphorylation on serine 1981 and phosphorylation of histone H2AX on serine 139 (γH2AX) are the key events reporting DNA damage, primarily formation of DNA double strand breaks. These events are detected immunocytochemically in individual cells using phospho-specific Abs. The protocols are presented that describe the methodology of immunofluorescent labeling of cells in conjunction with specific staining of cellular DNA. Flow- and imaging-Cytometry, the latter exemplified as Laser Scanning Cytometry, is used to quantify intensity of cellular fluorescence reporting activation of ATM and induction of γH2AX with respect to cellular DNA content, which in turn reports the cell cycle phase. Different protocols are presented for analysis of cells either grown in suspension or attached to surface of culture vessels. Examples of ATM activation and H2AX phosphorylation in response to DNA damage in leukemic HL-60 cells by DNA topoisomerase I inhibitor topotecan, and in lung carcinoma A549 cells by hydrogen peroxide, are presented.
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Laser Scanning Cytometry principles and applications an update
Methods of Molecular Biology, 2013Co-Authors: Piotr Pozarowski, Elena Holden, Zbigniew DarzynkiewiczAbstract:Laser Scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow Cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM.
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Laser Scanning Cytometry for automation of the micronucleus assay
Mutagenesis, 2011Co-Authors: Zbigniew Darzynkiewicz, Maxime François, Wayne R. Leifert, Ed Luther, Elena Holden, Piotr Smolewski, Mel Henriksen, Michael FenechAbstract:Laser Scanning Cytometry (LSC) provides a novel approach for automated scoring of micronuclei (MN) in different types of mammalian cells, serving as a biomarker of genotoxicity and mutagenicity. In this review, we discuss the advances to date in measuring MN in cell lines, buccal cells and erythrocytes, describe the advantages and outline potential challenges of this distinctive approach of analysis of nuclear anomalies. The use of multiple Laser wavelengths in LSC and the high dynamic range of fluorescence and absorption detection allow simultaneous measurement of multiple cellular and nuclear features such as cytoplasmic area, nuclear area, DNA content and density of nuclei and MN, protein content and density of cytoplasm as well as other features using molecular probes. This high-content analysis approach allows the cells of interest to be identified (e.g. binucleated cells in cytokinesis-blocked cultures) and MN scored specifically in them. MN assays in cell lines (e.g. the CHO cell MN assay) using LSC are increasingly used in routine toxicology screening. More high-content MN assays and the expansion of MN analysis by LSC to other models (i.e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure.
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analysis of cellular dna content by flow and Laser Scanning Cytometry
Advances in Experimental Medicine and Biology, 2010Co-Authors: Zbigniew Darzynkiewicz, Dorota H Halicka, Hong ZhaoAbstract:This chapter covers several aspects of methodology of DNA content analysis in individual cells that is most commonly used for assessment of DNA ploidy and for enumeration of cells in particular phases of the cell cycle. Briefly presented are general principles of instrumentation and cell analysis by flow- and Laser Scanning- Cytometry. Described are major methods designed to stain DNA with fluorochromes in live cells, in detergent-permeabilized cells, in cells fixed prior to DNA staining as well as in nuclei of cells isolated from paraffin-embedded tissues. Briefly addressed are approaches to estimate cellular DNA content in conjunction with cellular immunophenotype. Discussed are factors that affect accuracy of DNA content measurement such as: (i) differences in chromatin structure of the analyzed cells that restrict DNA accessibility to fluorochromes, (ii) stoichiometry of interaction between fluorochromes and DNA in chromatin and (iii) chemical mass action law defining dependency of fluorochrome binding to DNA in relation to fluorochrome concentration and number of potential binding sites in a sample. Described also are controls used to ensure accuracy of DNA ploidy determination, the principles in ploidy assessment and possible pitfalls in analysis.
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dna damage response induced by tobacco smoke in normal human bronchial epithelial and a549 pulmonary adenocarcinoma cells assessed by Laser Scanning Cytometry
Cytometry Part A, 2009Co-Authors: Hong Zhao, Anthony P Albino, Ellen D Jorgensen, Frank Traganos, Zbigniew DarzynkiewiczAbstract:Cigarette smoke (CS) is a major cause of lung cancer and a contributor to the development of a wide range of other malignancies. There is an acute need to develop a methodology that can rapidly assess the potential carcinogenic properties of the genotoxic agents present in CS. We recently reported that exposure of normal human bronchial epithelial cells (NHBEs) or A549 pulmonary carcinoma cells to CS induces the activation of ATM through its phosphorylation on Ser1981 and phosphorylation of histone H2AX on Ser139 (γH2AX) most likely in response to the formation of potentially carcinogenic DNA double-strand breaks (DSBs). To obtain a more complete view of the DNA damage response (DDR) we explored the correlation between ATM activation, H2AX phosphorylation, activation of Chk2 through its phosphorylation on Thr68, and phosphorylation of p53 on Ser15 in NHBE and A549 cell exposed to CS. Multiparameter analysis by Laser Scanning Cytometry made it possible to relate these DDR events, detected immunocytochemically, with cell cycle phase. The CS-dose-dependent induction and increase in the extent of phosphorylation of ATM, Chk2, H2AX, and p53 were seen in both cell types. ATM and Chk2 were phosphorylated ∼1 h prior to phosphorylation of H2AX and p53. The dephosphorylation of ATM, Chk2, and H2AX was seen after 2 h following CS exposure. The dose-dependency and kinetics of DDR were essentially similar in both cell types, which provide justification for the use of A549 cells in the assessment of genotoxicity of CS in lieu of normal bronchial epithelial cells. The observation that DDR was more pronounced in S-phase cells is consistent with the mechanism of induction of DSBs occurring as a result of collision of replication forks with primary lesions such as DNA adducts that can be caused by CS-generated oxidants. The cytometric assessment of CS-induced DDR provides a means to estimate the genotoxicity of CS and to explore the mechanisms of the response as a function of cell cycle phase and cell type. © 2009 International Society for Advancement of Cytometry
Louis A Kamentsky - One of the best experts on this subject based on the ideXlab platform.
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next generation Laser Scanning Cytometry
Methods in Cell Biology, 2004Co-Authors: Ed Luther, Louis A Kamentsky, Melvin Henriksen, Elena HoldenAbstract:Publisher Summary Flow Cytometry (FC) has been at the forefront of quantitative cytometric analysis. Recent experimental needs in the life sciences demand a combination of quantitative Cytometry and imaging Cytometry. This demand has been fulfilled by the development of Laser Scanning Cytometry (LSC). LSC is a combination of quantitative Cytometry and imaging Cytometry. LSC technology transforms the microscope from a qualitative to a quantitative tool for cell biology. Laser Scanning cytometer and two newer, next-generation systems, the automated imaging cytometer (iCyte), and the research imaging cytometer (iCys) are a product line of Laser Scanning cytometers. The iCys and iCyte systems provide for either interactive (iCys) or walkaway (iCyte) analysis. The chapter gives an overview of obtaining the images; segmentation and feature extraction; and data analysis for these cytometers. The iNovator application development module adds significantly to the capabilities of the iCyte and iCys systems. With the iNovator, the user can (1) employ imaging tools to the segmentation and data analysis process, (2) control the process with visually oriented macros, and (3) perform multiscale Scanning and analysis. The user has the ability to define and save numerous types of data files, both numerical and image. A number of applications have been developed for the new iCys and iCyte platforms.
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Laser Scanning Cytometry
Methods in Cell Biology, 2001Co-Authors: Louis A KamentskyAbstract:Publisher Summary Laser Scanning Cytometry (LSCM) automatically measures Laser excited fluorescence at multiple wavelengths and light scatter from cells on slides that have been treated with one or more fluorescent dyes to rapidly determine multiple cellular constituents and other features of the cells. This chapter describes a specific Laser Scanning cytometer, the LSC that can use these techniques perfected for flow Cytometry (FCM) to provide data comparable to FCM. Because it is microscope based and measures cells on the surface of a slide, records position of each cell on the slide, and has higher resolution, it can provide a number of benefits that may make it a more suitable cytometer for certain applications. LSCM is not comparable to confocal microscopy. Because LSCM must uniformly illuminate cells throughout their volume to obtain accurate whole cell constituent measurements, its optical components are designed to be nonconfocal. LSCM uses large field depths, and confocal microscopy emphasizes short field depth to provide detailed images at a narrow depth focal plane through each cell that is imaged. Additionally, LSCM is designed to automatically measure large heterogeneous populations of cells, unlike the detailed single cell analysis, for which confocal microscopy is most useful.
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multiparameter analysis of progeny of individual cells by Laser Scanning Cytometry
Cytometry, 2000Co-Authors: Elzbieta Bedner, Louis A Kamentsky, Qin Ruan, Sophie Chen, Zbigniew DarzynkiewiczAbstract:Background Effectiveness of antitumor drugs to suppress unrestricted proliferation of cancer cells is commonly measured by cell clonogenicity assays. Assays of clonogenicity are also used in studies of stem/progenitor cells and in analysis of carcinogenic transformation. The conventional assays are limited to providing information about frequency of colonies (cloning efficiency) and do not reveal the qualitative (phenotype) attributes of individual colonies that may yield clues on mechanisms by which cell proliferation was affected by the studied agent. Methods Laser Scanning Cytometry (LSC) was adapted to identify and characterize size and phenotype of colonies of MCF-7 cells growing in microscope slide chambers, untreated and treated with the cytotoxic ribonuclease, onconase (Onc). Individual colonies were located and data representing each colony were segmented based on >650-nm fluorescence excited by a He-Ne Laser of the cells whose protein was stained with BODIPY 630/650-X. The DNA of the cells was stained with propidium iodide (red fluorescence) whereas specific proteins (estrogen receptor [ER] or tumor suppressor p53) were detected immunocytochemically (green fluorescence), each excited by an Ar ion Laser. Results A plethora of attributes of individual colonies were measured, such as (a) morphometric features (area, circumference, area/circumference ratio, DNA or protein content per area ratio), (b) number of cells (nuclei), (c) DNA content, (d) protein content and protein/DNA ratio, and (e) expression of ER or p53 per colony, per total protein, per nucleus or per DNA, within a colony. Also cell cycle distribution within individual colonies and heterogeneity of colonies with respect to all the measured features could be assessed. The colonies growing in the presence of Onc had many of the above attributes different than the colonies from the untreated cultures. Conclusions Analysis of the features of cell colonies by LSC provides a wealth of information about the progeny of individual cells. Changes in colony size and phenotype, reflecting altered cell shape, cell size, colony protein/DNA ratio, and expression of individual proteins, may reveal mechanisms by which drugs suppress the proliferative capacity of the cells. This may include inducing growth imbalance and differentiation and modulating expression of the genes that may be associated with cell cycle, apoptosis, or differentiation in a progeny of individual cells. Extensions of LSC may make it applicable for automatic analysis of cloning efficiency and multiparameter analysis of cell colonies in soft agar. Such analyses may be useful in studies of the mechanisms and effectiveness of antitumor drugs, in the field of carcinogenesis, and for analyzing primary cultures and assessing tumor prognosis and drug sensitivity. The assay can also be adapted to analysis of microbial colonies. Cytometry 40:271–279, 2000 © 2000 Wiley-Liss, Inc.
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multiparameter analysis of dna content and cytokeratin expression in breast carcinoma by Laser Scanning Cytometry
Archives of Pathology & Laboratory Medicine, 1997Co-Authors: Richard J. Clatch, James R. Foreman, Jami L Walloch, Louis A KamentskyAbstract:Objective The objective of this study was to test a new laboratory technology, Laser Scanning Cytometry, for the purpose of performing multiparameter DNA content analysis of breast carcinomas. Design We developed a simplified method of multiparameter DNA content analysis using cytokeratin expression to positively gate epithelial cells. Over 300 consecutive cases of breast carcinoma were analyzed by multiparameter Laser Scanning Cytometry. The first 73 cases were analyzed in parallel by single parameter flow Cytometry. Setting The Department of Pathology, Christ Hospital and Medical Center, Oak Lawn, Ill. Specimens Three hundred eighteen consecutive cases of breast carcinoma presenting between March 1994 and December 1995. Main outcome measures Outcome measures included the percentage of cases for which DNA content analysis could be successfully performed given the limitations of specimen size. Additionally, for the first 73 cases, Laser Scanning Cytometry results were compared with flow Cytometry results. Results All of the first 73 cases were successfully analyzed by Laser Scanning Cytometry, but for 8 cases (11%) there was insufficient material for flow Cytometry. Correlation of DNA content for the remaining 65 cases analyzed in parallel by the two methods was nearly perfect (p = .994). Five seemingly discrepant cases highlighted the importance of cytokeratin gating of epithelial cells by any technique, as well as other advantages specific to Laser Scanning Cytometry, such as the ability to examine individual cells microscopically and correlate cytologic morphology with DNA content results. Conclusions Laser Scanning Cytometry is a promising new technology for DNA content analysis of solid tissue tumors. Further work needs to be performed to validate the prognostic potential of the Laser Scanning cytometric assay results and to generate methodologies aimed at providing highly objective determinations of tumor cell S-phase fraction.
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slide based Laser Scanning Cytometry
Acta Cytologica, 1997Co-Authors: Louis A Kamentsky, Douglas E Burger, Russell J Gershman, Ed LutherAbstract:OBJECTIVE: To show that Laser Scanning Cytometry (LSCM) can provide data equivalent to flow Cytometry (FCM) data and furnish a number of benefits, including cell relocation for visualization and several additional measurement features that may make it more suitable than FCM for pathology laboratories. STUDY DESIGN: A Laser Scanning cytometer, the LSC, was developed. Several instruments, at sites in the United States and Japan during the last two years, provided data characterizing the instrument and its usefulness. RESULTS: Data describing the sensitivity, precision, accuracy, utility of added measurement features and cell relocation capabilities of the LSC are presented. The data illustrate the applicability of the LSC to multiparameter DNA ploidy studies, resolution of phases of the cell cycle and cytogenetics. CONCLUSION: Because it is microscope based and measures cells on a slide, not in a flow chamber; records the position of each cell on the slide; and has higher resolution, LSCM provides a number of benefits that may make it more suitable than FCM for pathology laboratories.
Elzbieta Bedner - One of the best experts on this subject based on the ideXlab platform.
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use of flow and Laser Scanning Cytometry to study mechanisms regulating cell cycle and controlling cell death
Hematology-oncology Clinics of North America, 2002Co-Authors: Zbigniew Darzynkiewicz, Piotr Smolewski, Elzbieta BednerAbstract:This article presents major applications of flow and Laser Scanning Cytometry in studies of cell cycle and cell neurobiology. Described are representative examples of multivariate analysis of different attributes of the cell that reveal correlations between cellular DNA content; DNA replication; and expression of cyclins D1-3, E, A, and B1. Also described are major applications of Cytometry in studies of apoptosis, specifically in measurement of Bax translocation to mitochondria and nuclear factor kappa B to the nucleus, and in detection of DNA fragmentation.
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liquidless cell staining by dye diffusion from gels and analysis by Laser Scanning Cytometry potential application at microgravity conditions in space
Cytometry, 2001Co-Authors: Piotr Smolewski, Elzbieta Bedner, Wojciech Gorczyca, Zbigniew DarzynkiewiczAbstract:Background Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable—e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. Methods The “liquidless” staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by Laser Scanning Cytometry (LSC). Results Satisfactory cell and tissue staining, with minimal background, was achieved after 10–20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2–4-fold higher than the concentrations used routinely in Cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye—e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in “aqueous” medium was satisfactory. Conclusions Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions. Cytometry 44:355–360, 2001. © 2001 Wiley-Liss, Inc.
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detection of caspases activation by fluorochrome labeled inhibitors multiparameter analysis by Laser Scanning Cytometry
Cytometry, 2001Co-Authors: Piotr Smolewski, Elzbieta Bedner, Tzechen Hsieh, David J Phelps, Zbigniew DarzynkiewiczAbstract:Background The fluorochrome-labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308–313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and Laser Scanning Cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. Methods Apoptosis of human MCF-7, U-937, or HL-60 cells was induced by camptothecin (CPT) or tumor necrosis factor-α (TNF-α) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA-specific fluorochromes was measured by LSC. Results Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF-7 cells treated with CPT. FLICA binding was largely (>90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. Conclusions The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation. Cytometry 44:73–82, 2001. © 2001 Wiley-Liss, Inc.
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chapter 4 use of flow and Laser Scanning Cytometry in analysis of cell death
Methods in Cell Biology, 2001Co-Authors: Zbigniew Darzynkiewicz, Elzbieta BednerAbstract:Publisher Summary This chapter discusses the use of flow and Laser-Scanning Cytometry in analysis of cell death. It discusses that flow Cytometry is often used to measure cellular levels of the immunocytochemically-detected components that are involved directly or indirectly in the regulation and/or execution of apoptosis. The most prominent among them are members of the Bcl-2 protein family, caspases, the protooncogenes, or tumor suppressor genes. Flow Cytometry is widely used to study functional attributes of the cell, such as mitochondrial metabolism, oxidative stress, intracellular pH, or ionized calcium, all closely associated with mechanisms regulating cell sensitivity to apoptosis. The major advantage of flow Cytometry in these applications is that it offers the possibility of multiparametric measurements of a multitude of cell attributes. The second group of Cytometry applications comprises the methods to identify and quantify dead cells and discriminate between apoptotic vs necrotic modes of death. The chapter discusses the plethora of methods developed, especially for the identification of apoptotic cells. A Laser-Scanning cytometer (LSC) is a microscope-based cytofluorometer manufactured, which offers the combined advantages of flow Cytometry and image analysis LSC measures cell fluorescence rapidly and with accuracy comparable to that obtained by flow Cytometry.
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analysis of human tumors by Laser Scanning Cytometry
Methods in Cell Biology, 2001Co-Authors: Wojciech Gorczyca, Elzbieta Bedner, Myron R Melamed, A Deptala, Zbigniew DarzynkiewiczAbstract:Publisher Summary The Laser Scanning cytometer (LSC) is a microscope-based cytofluorometer that combines advantages of flow Cytometry and image analysis. Fluorescence of individual cells is measured rapidly by LSC, with sensitivity and accuracy comparable to that of flow Cytometry (FC). The unique feature of LSC, which distinguishes it from FC, is that the specimen is interrogated by a single or two Laser beams while located on a microscope slide rather than in suspension. Several attributes can be measured and recorded by LSC for each analyzed cell in a given cellular specimen: (1) the integrated fluorescence intensity over the integration contour at a given wavelength band, (2) the value of maximal pixel within the measured area, and (3) the perimeter of the contour. This chapter focuses on the clinical applications of LSC for tumor analysis. Several analytical methods, frequently used in FC for tumor diagnosis and prognosis, have been adapted to LSC and are presented in the chapter. The analytical power of LSC combined with its unique advantages, in particular to correlate measurements with visual examination of the corresponding cell, are of special value for pathologists.
Richard J. Clatch - One of the best experts on this subject based on the ideXlab platform.
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simplified immunophenotypic analysis by Laser Scanning Cytometry
Cytometry, 1998Co-Authors: Richard J. Clatch, James R. Foreman, Jami L WallochAbstract:Immunophenotypic analysis of hematologic specimens is a useful laboratory adjunct to surgical pathology and cytology to confirm or further characterize diagnoses of leukemia or lymphoma. Laser Scanning Cytometry is a new laboratory technology that has been adapted to perform immunophenotypic analysis of hematologic specimens, with numerous advantages as compared with flow Cytometry. In order to make full use of the Laser Scanning cytometer's capabilities, a new method of specimen preparation and means of performing the immunofluorescent reactions was developed. The technique described in this report, specific only to Laser Scanning Cytometry, enables panels of up to 36 different antibodies to be used on specimens as small as 50,000 total cells. The laboratory methodology is simple, requires 85% less antibody than flow cytometric methods, and allows individual cell cytologic morphology to be correlated with objective physical and fluorescent measurements on a cell-by-cell basis. Other advantages are described in the text. Over the course of nine months in our community hospital, we have used this technique clinically to analyze 172 cases of suspected leukemia or lymphoma. The method has proven remarkably useful, particularly for extremely small specimens such as fine needle aspiration biopsies.
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Five-color immunophenotyping plus DNA content analysis by Laser Scanning Cytometry
Communications in Clinical Cytometry, 1998Co-Authors: Richard J. Clatch, James R. ForemanAbstract:Laser Scanning Cytometry is a new laboratory technology similar to flow Cytometry but with advantages for certain clinical and research applications. To date, Laser Scanning Cytometry has been successfully used to perform three-color immunophenotypic analysis of hematologic specimens, single-color immunophenotyping plus DNA content analysis of numerous specimen types, and automated analysis of fluorescence in situ hybridization specimens. Several other interesting applications are also in development. In general, advantages of Laser Scanning Cytometry include reduced specimen size requirements, simplified methodologies, and the ability to microscopically examine individual cells-allowing for the direct correlation of cytologic morphology with objective fluorescence measurements. In this report, we describe a method which more fully takes advantage of the Laser Scanning cytometer's capabilities for immunophenotypic analysis of hematologic specimens. Specifically, we have devised a method to increase the number of fluorescent parameters from three to a total of six, five representing binding of immunofluorescent antibodies and one for stoichiometric measurements of DNA content. As with most Laser Scanning cytometric applications, this technique can be utilized on extremely small specimens and enables direct correlation of all of the measured fluorescent parameters with light microscopic cytologic morphology
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multiparameter analysis of dna content and cytokeratin expression in breast carcinoma by Laser Scanning Cytometry
Archives of Pathology & Laboratory Medicine, 1997Co-Authors: Richard J. Clatch, James R. Foreman, Jami L Walloch, Louis A KamentskyAbstract:Objective The objective of this study was to test a new laboratory technology, Laser Scanning Cytometry, for the purpose of performing multiparameter DNA content analysis of breast carcinomas. Design We developed a simplified method of multiparameter DNA content analysis using cytokeratin expression to positively gate epithelial cells. Over 300 consecutive cases of breast carcinoma were analyzed by multiparameter Laser Scanning Cytometry. The first 73 cases were analyzed in parallel by single parameter flow Cytometry. Setting The Department of Pathology, Christ Hospital and Medical Center, Oak Lawn, Ill. Specimens Three hundred eighteen consecutive cases of breast carcinoma presenting between March 1994 and December 1995. Main outcome measures Outcome measures included the percentage of cases for which DNA content analysis could be successfully performed given the limitations of specimen size. Additionally, for the first 73 cases, Laser Scanning Cytometry results were compared with flow Cytometry results. Results All of the first 73 cases were successfully analyzed by Laser Scanning Cytometry, but for 8 cases (11%) there was insufficient material for flow Cytometry. Correlation of DNA content for the remaining 65 cases analyzed in parallel by the two methods was nearly perfect (p = .994). Five seemingly discrepant cases highlighted the importance of cytokeratin gating of epithelial cells by any technique, as well as other advantages specific to Laser Scanning Cytometry, such as the ability to examine individual cells microscopically and correlate cytologic morphology with DNA content results. Conclusions Laser Scanning Cytometry is a promising new technology for DNA content analysis of solid tissue tumors. Further work needs to be performed to validate the prognostic potential of the Laser Scanning cytometric assay results and to generate methodologies aimed at providing highly objective determinations of tumor cell S-phase fraction.
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multiparameter immunophenotypic analysis of fine needle aspiration biopsies and other hematologic specimens by Laser Scanning Cytometry
Acta Cytologica, 1997Co-Authors: Richard J. Clatch, Jami L WallochAbstract:OBJECTIVE: To test the new laboratory technology of Laser Scanning Cytometry with respect to immunophenotyping of all types of hematologic and lymphoreticular specimens and particularly those of limited size, such as fine needle aspiration biopsies and hypocellular body fluids. STUDY DESIGN: Over the course of two years, 343 hematologic and lymphoreticular specimens of all types were immunophenotyped by Laser Scanning Cytometry using methodologies modified from those of conventional flow cytometric immunophenotyping. Results for all cases were corroborated with histology and/or cytology and, for some cases, immunohistochemistry and/or flow cytometric immunophenotyping. RESULTS: Over 98% of the 343 cases were successfully immunophenotyped by Laser Scanning Cytometry. These included many hypocellular specimens, such as 38 fine needle aspiration biopsies and 33 body fluid specimens. CONCLUSION: Laser Scanning Cytometry is a new laboratory technology with several significant advantages relative to flow Cytometry for immunophenotypic analysis of hematologic malignancy. The laboratory techniques are simplified, and antibody usage is reduced by 80%. Even more important, full-panel immunophenotyping with multiple antibodies can be performed on specimens as small as 50,000 cells total, making the technology particularly relevant to cytopathology. After immunophenotypic analysis, specimens can be stained for light microscopic examination, and individual cells meeting user-defined antigenic or physical characteristics can be automatically relocalized.
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immunophenotypic analysis of hematologic malignancy by Laser Scanning Cytometry
American Journal of Clinical Pathology, 1996Co-Authors: Richard J. Clatch, Jami L Walloch, Mary M Zutter, Louis A KamentskyAbstract:The authors tested a newly-developed computerized Laser Scanning cytometer (LSC) as a means of performing immunophenotypic analysis of hematologic specimens within their community hospital. Results were compared on a case-by-case basis with parallel flow cytometric and immunohistochemical data. A total of 71 specimens analyzed include 22 excised lymph nodes or other tissue biopsies, 18 peripheral bloods, 17 bone marrow aspirates, 7 body fluids, and 7 fine-needle aspiration biopsies of lymphoid tissue. The LSC proved to be a useful instrument capable of generating simultaneous two-color immunofluorescent data directly analogous to that obtained via conventional flow Cytometry. However, Laser Scanning cytometric analysis provides advantages over flow cytometric analysis, because the LSC measures cells on a slide rather than in a fluid stream. Specifically, cells can be microscopically examined at any time--before, during, or after automated immunofluorescent analysis. In addition, specimen preparation techniques are less restricted and more cost efficient. Lastly, even extremely small and/or hypocellular specimens (such as body fluids and fine-needle aspiration biopsies) can be successfully analyzed.
Attila Tarnok - One of the best experts on this subject based on the ideXlab platform.
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Laser Scanning Cytometry in human brain slices
Cytometry Part A, 2006Co-Authors: Birgit Mosch, Anja Mittag, Dominik Lenz, Thomas Arendt, Attila TarnokAbstract:Background The Laser Scanning Cytometry (LSC) offers quantitative fluorescence analysis of cell suspensions and tissue sections. Methods We adapted this technique to immunohistochemical labelled human brain slices. Results We were able to identify neurons according to their labelling and to display morphological structures such as the lamination of the entorhinal cortex. Further, we were able to distinguish between neurons with and without cyclin B1 expression and we could assign the expression of cyclin B1 to the cell islands of layer II and the pyramidal neurons of layer V of the entorhinal cortex in Alzheimer's disease effected brain. In addition, we developed a method depicting the three-dimensional distribution of the cells in intact tissue sections. Conclusions In this pilot experiments we could demonstrate the power of the LSC for the analysis of human brain sections. © 2006 International Society for Analytical Cytology
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sequential photobleaching for increasing the measurable fluorochromes by Laser Scanning Cytometry
Biomedical optics, 2004Co-Authors: Anja Mittag, Dominik Lenz, O Andreas H M D Gerstner, Ulrich Sack, Jozsef Bocsi, Attila TarnokAbstract:For immunophenotypic analysis more measurable parameters for the discrimination of leukocyte subsets are necessary. With a single scan six fluorochromes can be distinguished with the Laser Scanning Cytometer (LSC). Due to the number of PMTs the amount of simultaneously measurable fluorescences per scan is limited. Nevertheless, the amount of measurable colors can be improved to eight by appropriate change of the filter settings and two scans per specimen. Aim of this study was to use the special features of Slide based Cytometry (SBC) beyond filter change, remeasurement and merging to distinguish fluorochromes with similar emission spectra. The photosensitivity of fluorochromes that are excited and emit in a similar wavelength range may be very different. The number of measurable parameters per PMT was increased using photosensitivity of different fluorochromes as additional criteria. Peripheral blood leukocytes were stained with antibodies conjugated to the fluorochromes APC, APC-Cy5.5 and Alexa-Fluor 633 and mounted on conventional uncoated glass slides with Fluorescence mounting medium. Specimens were excited in the LSC with the HeNe (633nm) Laser and measured at different filter settings (670/20nm-filter for APC/ALEXA 633 and 710/20nm-filter for APC-Cy5.5). At this point, APC-Cy5.5 and APC/ALEXA633 were already distinguishable. In order to differentiate between APC and ALEXA633 photobleaching was performed by repeated excitation with the Laser at 633nm. Control measurements proved that APC is much more sensitive against Laser excitation, i.e. looses much more fluorescence intensity than ALEXA633. The separate measurements (before/after filter change and before/after bleaching) were merged into one file. The photostability of Alexa-Fluor 633 (1.02% bleach per scan) and APC (5.74% bleach per scan) are substantially different. Therefore, after bleaching and merging both fluorochromes can be distinguished and are regarded by the software as separate parameters. The fluorochromes APC/ALEXA633 and APC-Cy5.5 can be discriminated by changing the emission filters before bleach. By sequential photobleaching , change of filters and subsequent merging of the data the number of simultaneously measurable “colors” is substantially increased.
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six and more color immunophenotyping on the slide by Laser Scanning Cytometry lsc
Biomedical optics, 2003Co-Authors: Dominik Lenz, W Laffers, F Bootz, O Andreas H M D Gerstner, Michae Steinbrecher, Attila TarnokAbstract:ABSTRACT The request for a more profound immunophenotyping and sometimes the lack of material demands more measurablefluorescence colors to increase the number of detectable antigens per specimen. Six different fluorescences are distin-guishable in the Laser Scanning Cytometer (LSC) 1 . In the present study we wanted to increase this number to eightcolors per measurement. Combined with an earlier study it is likely possible to measure n fluorescences i.e. n leuko-cyte subsets by a series of measurements followed by subsequent restaining steps. The new method is realized by s-ing the combination of filter change and a subsequent re-measurement for the distinction between the fluorescent dyesCy5 and Cy5.5. The optical filters are replaced after the first measurement and the same specimen is remeasuredwithout removing it from the microscope. For the sec ond measurement a filter is inserted that detects Cy5.5 but notCy5 (710/10nm). After the second meas urement of the same specimen both data files are combined. With the aid ofthis feature it is possible to line out the differences between both measurements. If the data from the second measu r-ing (Cy5.5 only) is subtracted from the first, Cy5 data is the result. After the first two measurements when eight di f-ferent fluorescences (i.e. antigens or leukocyte subsets) were analyzed, the same cells are restained and a new meas-urement is performed. In theory, one can perform n re-measurements with eight fluorescences respectively. The in-formation gained per specimen is only limited by the number of available antibodies and b sterical hindrance. Keywords: immunophenotyping, Laser Scanning Cytometry, slide-based Cytometry, Cy5.5, tandem dyes
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analysis of ploidy in hypopharyngeal cancer by Laser Scanning Cytometry on fine needle aspirate biopsies
Analytical cellular pathology : the journal of the European Society for Analytical Cellular Pathology, 2003Co-Authors: Andreas O H Gerstner, F Bootz, Julia Machlitt, Hansjurgen Welkoborsky, Attila TarnokAbstract:Aim To test Laser Scanning Cytometry (LSC) for the analysis of ploidy in squamous cell carcinoma of the hypopharynx (SCCH) and to develop a routine application for minimal samples such as fine needle aspirate biopsies (FNABs). Methods: From 11 individuals 30 FNABs of primary tumors (n=11) and lymphatic metastases of SCCH (n=11) and non‐metastatic lymph nodes (n=8) are analyzed by LSC. This microscope based instrument scans the cells after immobilization on a glass slide and after double staining of cytokeratin and DNA. The location of each cell is stored with the fluorescence data. Therefore the morphology of every cell can be documented by re‐staining with H&E and re‐localization on the slide. Additionally, aliquots are Feulgen‐stained for image Cytometry in 8 specimens. Results: The diploid reference peak is identified taking leukocytes as internal standard. The DNA‐index of the carcinoma cells ranges from 0.4 to 3.8. Comparison with image Cytometry shows good correlation (r=0.89). Conclusion: LSC provides a reliable and objective way to determine the ploidy of SCCH pre‐operatively. Colour figures can be viewed on http://www.esacp.org/acp/2003/25‐2/gerstner.htm.
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near infrared dyes for six color immunophenotyping by Laser Scanning Cytometry
Cytometry, 2002Co-Authors: Andreas O H Gerstner, W Laffers, F Bootz, Dominik Lenz, Robert A Hoffman, Michael Steinbrecher, Attila TarnokAbstract:Background To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the Laser Scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. Methods The optical filters of the LSC were replaced—photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. Results With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon Laser) or 633-nm (helium-neon Laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. Conclusions With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC. Cytometry 48:115–123, 2002. © 2002 Wiley-Liss, Inc.
