Cytometry. Part A : the journal of the International Society for Analytical Cytology最新文献

{"title":"Segmentation of complex cell clusters in microscopic images: application to bone marrow samples.","authors":"Björn Nilsson,&nbsp;Anders Heyden","doi":"10.1002/cyto.a.20153","DOIUrl":"https://doi.org/10.1002/cyto.a.20153","url":null,"abstract":"<p><strong>Background: </strong>Morphologic examination of bone marrow and peripheral blood samples continues to be the cornerstone in diagnostic hematology. In recent years, interest in automatic leukocyte classification using image analysis has increased rapidly. Such systems collect a series of images in which each cell must be segmented accurately to be classified correctly. Although segmentation algorithms have been developed for sparse cells in peripheral blood, the problem of segmenting the complex cell clusters characterizing bone marrow images is harder and has not been addressed previously.</p><p><strong>Methods: </strong>We present a novel algorithm for segmenting clusters of any number of densely packed cells. The algorithm first oversegments the image into cell subparts. These parts are then assembled into complete cells by solving a combinatorial optimization problem in an efficient way.</p><p><strong>Results: </strong>Our experimental results show that the algorithm succeeds in correctly segmenting densely clustered leukocytes in bone marrow images.</p><p><strong>Conclusions: </strong>The presented algorithm enables image analysis-based analysis of bone marrow samples for the first time and may also be adopted for other digital cytometric applications where separation of complex cell clusters is required.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. Part A : the journal of the International Society for Analytical Cytology","volume":" ","pages":"24-31"},"PeriodicalIF":3.7,"publicationDate":"2005-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cyto.a.20153","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40930485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging.","authors":"Daniela Bratosin,&nbsp;Laura Mitrofan,&nbsp;Carmen Palii,&nbsp;Jérôme Estaquier,&nbsp;Jean Montreuil","doi":"10.1002/cyto.a.20152","DOIUrl":"https://doi.org/10.1002/cyto.a.20152","url":null,"abstract":"<p><strong>Background: </strong>A highly sensitive, fast, and simple flow cytometric assay to assess human red blood cell (RBCs) viability and aging is reported.</p><p><strong>Methods: </strong>The assay described in this report is based on the use of acetoxymethyl ester of calcein (calcein-AM), a fluorescein derivative and nonfluorescent vital dye that passively crosses the cell membrane of viable cells and is converted by cytosolic esterases into green fluorescent calcein, which is retained by cells with intact membranes and inactive multidrug resistance protein. The loss of calcein can be easily determined by flow cytometry, and the cytosolic localization of esterases was demonstrated by spectrofluorometric analyses.</p><p><strong>Results: </strong>We found that RBCs incubated with Ca(2+), which induces a rapid and modulated self-death that shares several features with apoptosis (Bratosin et al., Cell Death Differ 2001;8:1143-1156), externalized phosphatidylserine and lost calcein staining and cytosolic adenosine triphosphate content. Double labeling using phycoerythrin-labeled annexin-V and calcein-AM showed that the decrease of esterase activity is an early event that precedes the externalization of phosphatidylserine residues. In addition, this assay allowed us to distinguish young and aged RBCs isolated by ultracentrifugation in a self-forming Percoll gradient and can be considered as a reliable marker of RBC aging.</p><p><strong>Conclusions: </strong>Calcein-AM assay may represent a wide application for assessing RBC viability, particularly in blood banks.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. Part A : the journal of the International Society for Analytical Cytology","volume":" ","pages":"78-84"},"PeriodicalIF":3.7,"publicationDate":"2005-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cyto.a.20152","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40930467","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell cycle kinetic dysregulation in HIV-infected normal lymphocytes.","authors":"David M Asmuth,&nbsp;Nan Wang,&nbsp;Ying Lu,&nbsp;Xiao-Dong Li,&nbsp;Lisa Reece,&nbsp;Nicholas H A Terry,&nbsp;Richard B Pollard,&nbsp;Mostafa Nokta,&nbsp;James F Leary,&nbsp;R Allen White","doi":"10.1002/cyto.a.20148","DOIUrl":"https://doi.org/10.1002/cyto.a.20148","url":null,"abstract":"<p><strong>Background: </strong>Viruses alter cellular gene transcription and protein binding at many steps critical for cell cycle regulation to optimize the milieu for productive infection. Reasoning that virus-host cell interactions would result in perturbations of cell cycle kinetics, measurement of the duration of the phases of the cell cycle in normal T lymphocytes infected with human immunodeficiency virus (HIV) was undertaken.</p><p><strong>Methods: </strong>Flow cytometric measurement of bromodeoxyuridine-labeled and DNA content-stained cells at multiple points through the cell cycle allowed estimation of the fraction of cells in each phase, the potential doubling-time, and the durations of S and G(2)/M phases. Separate analysis of the HIV(+) and HIV(-) populations within the infected cultures was performed based on intracellular, anti-HIV core p24 antibody labeling. A novel mathematical model, which accounted for cell loss, was developed to estimate cell cycle phases.</p><p><strong>Results: </strong>(a) S phase was prolonged in the HIV-1(SF2)-infected cells compared with control. (b) This delay in S phase was due to delay in the population of cells not expressing HIV-1 antigens (p24 negative). (c) Accumulation of cells in G(2)/M phase was confirmed in HIV-1-infected cultures and was proportional to the level of infection as measured by p24 fluorescent intensity. However, all mock and HIV-1-infected populations predicted to proceed through cell division demonstrated similar G(2)/M-phase durations. (c) Potential doubling times were longer in the infected cultures; in contrast, the p24(+) subpopulations accounted for this delay. This suggests an isolated delay in the G(0)/G(1) phase for that population of cells.</p><p><strong>Conclusions: </strong>Multiple phases of host cell cycle durations were affected by HIV-1(SF2) infection in this in vitro model, suggesting novel HIV-1 pathogenesis mechanisms. Prolonged S-phase durations in HIV-1 infected/p24(-) and G(0)/G(1)-phase durations in HIV-1 infected/p24(+) subpopulations require further study to identify mechanistic pathways.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. Part A : the journal of the International Society for Analytical Cytology","volume":" ","pages":"41-51"},"PeriodicalIF":3.7,"publicationDate":"2005-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cyto.a.20148","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40930487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Estimating cell death in G(2)M using bivariate BrdUrd/DNA flow cytometry.","authors":"R Allen White,&nbsp;David M Asmuth,&nbsp;Ying Lu,&nbsp;Nan Wang,&nbsp;Xiao-Dong Li,&nbsp;Lisa Reece,&nbsp;Richard B Pollard,&nbsp;Mostafa Nokta,&nbsp;James F Leary,&nbsp;Nicholas H A Terry","doi":"10.1002/cyto.a.20147","DOIUrl":"https://doi.org/10.1002/cyto.a.20147","url":null,"abstract":"<p><strong>Background: </strong>In an accompanying paper (Asmuth et al.) it was found necessary to include cell death explicitly to estimate parameters of cell proliferation. The use of bivariate flow cytometry to estimate the phase durations and the doubling times of cells labeled with thymidine analogues is well established. However, these methods of analysis do not consider the possibility of cell death. This report demonstrates that estimating cell death in G(2)/M is possible.</p><p><strong>Methods: </strong>Mathematical models for the experimental quantities, the fraction of labeled undivided cells, the fraction of labeled divided cells, and the relative movement were developed. These models include the possibility that, of the cells with G(2)/M DNA content, only a certain fraction will divide, with the remainder dying after some time T(R). Simulation studies were conducted to test the possibility of using simple methods to estimate phase durations and cell death rates.</p><p><strong>Results: </strong>Cell death alters the estimates of phase transit times in a rather complex manner that depends on the lifetime of the doomed cells. However, it is still possible to obtain estimates of the phase durations of cells in S and G(2)/M and the death rates of cells in G(2)/M.</p><p><strong>Conclusions: </strong>The methods presented herein provide a new way to characterize cell populations that includes cell death rates and common measurements of cell proliferation.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. Part A : the journal of the International Society for Analytical Cytology","volume":" ","pages":"32-40"},"PeriodicalIF":3.7,"publicationDate":"2005-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cyto.a.20147","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40930486","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bystander cell proliferation is modulated by the number of adjacent cells that were exposed to ionizing radiation.","authors":"Bogdan I Gerashchenko,&nbsp;Roger W Howell","doi":"10.1002/cyto.a.20150","DOIUrl":"https://doi.org/10.1002/cyto.a.20150","url":null,"abstract":"<p><strong>Background: </strong>Direct cell-to-cell contact appears to be a prerequisite for the proliferative response of bystander WB-F344 cells co-cultured with irradiated cells; however, neither gap junctional intercellular communication nor long-range factors released into the medium appear to be involved (Cytometry 2003;56A:71-80). The present work investigated whether the proliferative bystander response depends on the number of irradiated cells (cells exposed to external gamma-rays or cells exposed to short-range beta-particles emitted by DNA-incorporated (3)H-thymidine) that are adjacent to unirradiated bystander cells.</p><p><strong>Methods: </strong>Subconfluent monolayers of rat liver epithelial cells (WB-F344) were incubated in the presence of (methyl-(3)H)thymidine at a concentration of 5.8 kBq/ml for 18 h. Radiolabeled cells containing 0.7 x 10(-3) Bq/cell (absorbed dose: 0.14 Gy) were plated together with unlabeled cells in proportions of 6% and 94%, 12% and 88%, 25% and 75%, 50% and 50%, and 75% and 25%, respectively, keeping constant the total number of plated cells. In a parallel experiment, cells acutely exposed to 5 Gy of (137)Cs gamma-rays were plated with unirradiated cells in the same proportions. In both experiments, cells were co-cultured for 24 h followed by a flow cytometric study of their proliferation. The two cell populations in the co-cultures were distinguished by staining one population with carboxyfluorescein diacetate, succinimidyl ester, which metabolizes intracellularly.</p><p><strong>Results: </strong>Increasing the fraction of irradiated cells relative to unirradiated bystander cells led to an increase in proliferation of bystander cells. Specifically, in co-cultures in which irradiated cells were initially mixed with unirradiated cells in proportions of 50% and 50% and of 75% and 25%, respectively, bystander cells showed a statistically significant increase of their proliferation compared with the controls.</p><p><strong>Conclusions: </strong>The proliferative response of WB-F344 bystander cells is modulated by the number of adjacent cells that are exposed to ionizing radiation from external gamma-rays or intracellularly emitted (3)H beta-particles.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. Part A : the journal of the International Society for Analytical Cytology","volume":" ","pages":"62-70"},"PeriodicalIF":3.7,"publicationDate":"2005-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cyto.a.20150","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40930466","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Heterogeneity in DNA damage using the comet assay.","authors":"Peggy L Olive,&nbsp;Ralph E Durand","doi":"10.1002/cyto.a.20154","DOIUrl":"https://doi.org/10.1002/cyto.a.20154","url":null,"abstract":"<p><p>The single-cell gel electrophoresis or \"comet\" assay was developed many years ago to analyze DNA damage in individual cells. It is a powerful and versatile technique that relies on microscopic visualization or imaging of DNA after single cells are embedded in agarose, lysed, and electrophoresed. In addition, the basic methodology has been extended to permit the detection of a variety of classes of DNA damage with good sensitivity in virtually any single-cell type. A unique but understudied property of the comet assay is its ability to detect and quantify cellular heterogeneity in response to DNA-damaging agents. This review outlines the considerations in producing and analyzing comet data when heterogeneity in induction of or cellular response to DNA damage is the major consideration. Examples are presented to emphasize the heterogeneity of tumor response to ionizing radiation and cytotoxic drugs.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. Part A : the journal of the International Society for Analytical Cytology","volume":" ","pages":"1-8"},"PeriodicalIF":3.7,"publicationDate":"2005-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cyto.a.20154","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40936996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Automated image analysis methods for 3-D quantification of the neurovascular unit from multichannel confocal microscope images.","authors":"Gang Lin,&nbsp;Chris S Bjornsson,&nbsp;Karen L Smith,&nbsp;Muhammad-Amri Abdul-Karim,&nbsp;James N Turner,&nbsp;William Shain,&nbsp;Badrinath Roysam","doi":"10.1002/cyto.a.20149","DOIUrl":"https://doi.org/10.1002/cyto.a.20149","url":null,"abstract":"<p><strong>Background: </strong>There is a need for integrative and quantitative methods to investigate the structural and functional relations among elements of complex systems, such as the neurovascular unit (NVU), that involve multiple cell types, microvasculatures, and various genomic/proteomic/ionic functional entities.</p><p><strong>Methods: </strong>Vascular casting and selective labeling enabled simultaneous three-dimensional imaging of the microvasculature, cell nuclei, and cytoplasmic stains. Multidimensional segmentation was achieved by (i) bleed-through removal and attenuation correction; (ii) independent segmentation and morphometry for each corrected channel; and (iii) spatially associative feature computation across channels. The combined measurements enabled cell classification based on nuclear morphometry, cytoplasmic signals, and distance from vascular elements. Specific spatial relations among the NVU elements could be quantified.</p><p><strong>Results: </strong>A software system combining nuclear and vessel segmentation codes and associative features was constructed and validated. Biological variability contributed to misidentified nuclei (9.3%), undersegmentation of nuclei (3.7%), hypersegmentation of nuclei (14%), and missed nuclei (4.7%). Microvessel segmentation errors occurred rarely, mainly due to nonuniform lumen staining.</p><p><strong>Conclusions: </strong>Associative features across fluorescence channels, in combination with standard features, enable integrative structural and functional analysis of the NVU. By labeling additional structural and functional entities, this method can be scaled up to larger-scale systems biology studies that integrate spatial and molecular information.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. Part A : the journal of the International Society for Analytical Cytology","volume":" ","pages":"9-23"},"PeriodicalIF":3.7,"publicationDate":"2005-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cyto.a.20149","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40946451","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"New method to detect histone acetylation levels by flow cytometry.","authors":"Simona Ronzoni,&nbsp;Mario Faretta,&nbsp;Marco Ballarini,&nbsp;Piergiuseppe Pelicci,&nbsp;Saverio Minucci","doi":"10.1002/cyto.a.20151","DOIUrl":"https://doi.org/10.1002/cyto.a.20151","url":null,"abstract":"<p><strong>Background: </strong>Reversible histone acetylation affects chromatin structural organization, thus regulating gene expression and other nuclear events. Levels of histone acetylation are tightly modulated in normal cells, and alterations of their regulating mechanisms have been shown to be involved in tumorigenesis.</p><p><strong>Methods: </strong>We developed a new flow cytometric technique for detection of histone acetylation, based on a specific monoclonal antibody that recognizes acetylated histone tails. Bivariate analysis for histone acetylation levels and DNA were performed to study modulation of chromatin organization during the cell cycle and after induction of histone hyperacetylation by the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Histone acetylation and transcription levels were monitored during differentiation induced by retinoic acid alone or in combination with TSA. Blood samples from patients were analyzed with the described protocol to monitor the effects of HDAC inhibitors in vivo and validate the developed protocol for clinical usage.</p><p><strong>Results: </strong>Flow cytometric detection of acetylation status can successfully detect modifications induced by HDAC inhibitor treatment in vivo as demonstrated by analysis of various blood samples from patients treated with valproic acid. Changes in acetylation levels during the cell cycle demonstrated a reproducible increase in histone acetylation during the replication phase that was subsequently decreased at the G2M entrance, thus paralleling the behavior of DNA replication and transcriptional activity.</p><p><strong>Conclusions: </strong>Multiparameter analysis of histone acetylation and expression of molecular markers, DNA ploidy, and/or cell cycle kinetics can provide a quick and statistically reliable tool for the diagnosis and evaluation of treatment efficacy in clinical trials using HDAC inhibitors.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. Part A : the journal of the International Society for Analytical Cytology","volume":" ","pages":"52-61"},"PeriodicalIF":3.7,"publicationDate":"2005-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cyto.a.20151","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40930465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"D cyclins in lymphocytes.","authors":"David Kaplan,&nbsp;Howard Meyerson,&nbsp;William Husel,&nbsp;Kristine Lewandowska,&nbsp;Grayden MacLennan","doi":"10.1002/cyto.a.20103","DOIUrl":"https://doi.org/10.1002/cyto.a.20103","url":null,"abstract":"<p><strong>Background: </strong>D cyclins are essential for the progression of cells through the G1 phase of the cell cycle. There are three distinct D cyclins. Cyclin D1 has been shown to be expressed by many different types of cells but not by lymphocytes. Cyclins D2 and D3 have been found in lymphocytes.</p><p><strong>Methods: </strong>We used high-resolution enzymatic amplification staining technology in conjunction with flow cytometry and confocal microscopy and with immunoblotting to reassess the expression of the D cyclins in human lymphocytes.</p><p><strong>Results: </strong>Using high-resolution technology for flow cytometry, we found all three D cyclins in quiescent human peripheral blood lymphocytes. Cyclin D1 was expressed in quiescent and activated cells at levels commensurate with those of actively proliferating tumor cell lines. Cyclin D1 was functional inasmuch as it was complexed with CDK4. In the quiescent cells, cyclin D1 was expressed in the cytoplasm but, after activation, was found in the nucleus.</p><p><strong>Conclusions: </strong>These findings demonstrate that lymphocytes express cyclin D1 and necessitate a reappraisal of the hypothesis that the D cyclins subsume redundant activities with tissue-specific expression.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. Part A : the journal of the International Society for Analytical Cytology","volume":" ","pages":"1-9"},"PeriodicalIF":3.7,"publicationDate":"2005-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cyto.a.20103","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24878851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}