FuGEFlow: data model and markup language for flow cytometry

Background  

Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata.     

流式细胞术在乳酸菌自溶检测中的应用

【目的】使用流式细胞术(Flow Cytometric)建立一种新的检测方法,可快速筛选自溶度不同的乳酸菌菌株。【方法】菌悬液经20mmol/L的PI-PBS染液在4℃条件下避光染色30min,上流式细胞仪进行测定,检测器激发光波长488nm,检测波长630nm,每个样品收集1×105个细胞,联机使用CellQuest软件分析结果。【结果】阳性染色细胞数与细胞总数之比很好地反映菌液中自溶细胞与非自溶细胞的比例关系,整个检测过程耗时仅为1h左右。【结论】与传统检测方法比较,FCM测定结果稳定可靠,检测时间短,为乳酸菌的自溶特性研究及筛选自溶度不同的菌株用作商业发酵剂提供了便利条件。     

Systems biology and clinical cytomics: The 10th Leipziger Workshop and the 3rd International Workshop on Slide-Based Cytometry, Leipzig, Germany, April 2005.

Despite very significant technical and software improvements in flow cytometry (FCM) since the 1980's, the demand for a cytometric technology combining both quantitative cell analysis and morphological documentation in Cytomics became evident. Improvements in microtechnology and computing permit nowadays similar quantitative and stoichiometric single cell-based high-throughput analyses by microscopic instruments, like Slide-Based Cytometry (SBC). SBC and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures during early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance not only as a cytomics technology platform but also because of low quantities of required reagents and biological material. The exact knowledge of the location of each cell on the slide permits repetitive restaining and reanalysis of specimens. Various separate measurements of the same specimen can be ultimately fused to one database increasing the information obtained per cell. Relocation and optical evaluation of cells as typical SBC feature, can be of integral importance for cytometric analysis, since artifacts can be excluded and morphology of measured cells can be documented. Progress in cell analytic: In the SBC, new horizons can be opened by the new techniques of structural and functional analysis with the high resolution from intracellular and membrane (confocal microscopy, nanoscopy, total internal fluorescence microscopy (TIRFM), and tissue level (tissomics), to organ and organism level (in vivo cytometry, optical whole body imaging). Predictive medicine aims at the detection of changes in patient's state prior to the manifestation of the disease or the complication. Such instances concern immune consequences of surgeries or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Preventive anti-infectious or anti-shock therapy as well as curative chemotherapy in combination with stem cell transplantation may provide better survival chances for patient at concomitant cost containment. Predictive medicine-guided optimization of therapy could lead to individualized medicine that gives significant therapeutic effect and may lower or abrogate potential therapeutic side effects. The 10th Leipziger Workshop combined with the 3rd International Workshop on SBC aimed to offer new methods in Image- and Slide-Based Cytometry for solutions in clinical research. It moved towards practical applications in the clinics and the clinical laboratory. This development will be continued in 2006 at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.     

iPSCs来源的血细胞在血液系统疾病中的应用与发展

骨髓（BM）移植已在许多血液病中成功应用多年,主要依赖于成人BM和外周血（PB）中的造血干细胞（HSCs）。然而,合适配型HSCs的稀缺限制了其临床应用,难以满足更多有需要的患者。诱导多能干细胞（iPSCs）的出现,有望解决这一难题。iPSCs具有分化成所有血细胞的潜能,使得HSCs和其他血细胞在血液病中的广泛应用成为可能。然而,临床应用iPSCs治疗血液病仍障碍重重。本文主要回顾了iPSCs在血液病中的应用和发展以及面临的问题和挑战。     

Clinical applications of cytometry: 6th annual meeting.

The Sixth Annual Clinical Applications of Cytometry Meeting was held September 11-14, 1991, in Charleston, SC. Attendance reached a record 470. The meeting provides a forum for interactions among investigators who utilize cytometry as a tool in their clinical immunology, cell biology, hematology, and cancer investigations. Clinical laboratory directors and their technical staff find the meeting of practical value because of the presentation of new applications that they can take home to their own laboratories. The emphasis of the meeting is on advances in the application of cytometry to clinical problems. Often, advances result from new dyes or reagents or improved instrumentation. Sometimes they result from advances in biology that make the studies possible. Occasionally a new way of looking at the same data provides a useful answer. In every case, the effort is to provide a reliable, straightforward way to quantitate biologic information in order to provide improved diagnosis or treatment of human disease.     

Analysis of free intracellular calcium by flow cytometry: multiparameter and pharmacologic applications

Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca(2+) in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca(2+) in surface-marker-defined target cells as well as correlation of changes in Ca(2+) with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca(2+) by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca(2+) in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca(2+) with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca(2+) changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.     

Slide-based cytometry and predictive medicine: the 8th Leipziger workshop and the 1st international workshop on slide-based cytometry.

Slide-based cytometry (SBC) and related techniques offer unique tools to perform complex diagnostic procedures at very early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance, not only as a cytomics technology platform, but for patients with low blood volume such as neonates. The exact knowledge of the location of each cell on the slide allows the specimen to be restained and subsequently reanalyzed. These separate measurements can be fused to one data file (merging), increasing the information obtained per cell. Relocalization and optical evaluation of the cells, a typical feature of SBC, can be of integral importance for cytometric analysis. Predictive medicine is aimed at the detection of changes in the patient's state prior to the manifestation of deterioration or improvement. Such instances are concerned with multiorgan failure in sepsis or noninfectious posttraumatic shock in intensive care patients, or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Early anti-infectious or anti-shock therapy, as well as curative chemotherapy in combination with stem cell transplantation, may provide better survival chances for the patient as well as concomitant cost containment. Predictive medicine-guided, individualized, early reduction or cessation of therapy may lower or abrogate potential therapeutic side effects (individualized medicine). With the 8th Leipziger Workshop and the 1st International Workshop on Slide-Based Cytometry, cytomics technologies moved to more practical applications in the clinics and the clinical laboratory. This development will be continued in 2004, at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.     

幽门螺杆菌诱导人胃上皮细胞株BGC—823凋亡

目的和方法 :以Giemsa染色法、DNA条带分析、流式细胞仪分析和TUNEL法观察幽门螺杆菌 (Helicobacterpylor ,Hp)菌体超声粉碎提取物对人胃上皮细胞株BGC 82 3凋亡的影响。 结果 :四种方法的检测结果均显示Hp提取物能诱导BGC 82 3细胞凋亡 ,效应具有剂量依赖和时间依赖关系。结论 :Hp可能对胃粘膜细胞凋亡有直接的诱导作用。     

Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production

This paper describes a microalgal cell lipid fluorescence enhancement method using BODIPY(505/515), which can be used to screen for lipids in wild-type microalgae and to monitor lipid content within microalgae production processes to determine optimal harvesting time. The study was based on four microalgae species (Dunaliella teteriolecta, Tetraselmis suecica, Nannochloropsis oculata, and Nannochloris atomus) selected because of their inherent high lipid content. An extended analysis was carried out with N. oculata due to the depressed fluorescence observed when compared with the other experimental strains. BODIPY(505/515) lipid fluorescence was determined for two solvent pre-treatment methods (DMSO and glycerol) and four staining condition parameters (analysis time, staining temperature, dye concentration, and algal cell concentration). It was found that lipid fluorescence of thick cell-walled microalgae, such as N. oculata, is significantly enhanced by both the pre-treatment methods and staining condition parameters, thereby significantly enhancing lipid fluorescence by ca. 800 times the base autofluorescence. The lipid fluorescence enhancement method provides a quick and simple index for in vivo Flow Cytometry quantification of total lipid contents for purposes of species screening or whole culture monitoring in biofuel-directed microalgae production.     

Flow cytometric measurements of fluorescence energy transfer using single laser excitation

Flow cytometric energy transfer (FCET) measurements between labeled specific sites of cell surface elements (Sz?llosi et al., Cytometry, 5:210-216, 1984) have been extended in a simplified form using a flow cytometer equipped with single excitation beam. This versatile and easily applicable method has several advantages over any nonflow cytometric (i.e., spectrofluorimetric) energy transfer measurements on cell surfaces: The labeled ligands can be applied in excess, without washing, thereby enabling the investigation of relatively labile receptor-ligand complexes. Contributions of signals from cell debris, from cell aggregates, or from nonviable cells can be avoided by gating the data collection on the light scatter signal. The heterogeneity of the cell population with respect to the proximity of the labeled binding sites can be studied. In the cases of homologous ligands or of ligands binding to the same molecule but at different epitopes, the determination of fluorescence resonance energy transfer efficiency values can be carried out on a cell-by-cell basis, offering data on intramolecular conformational changes. This modified FCET method enabled us to demonstrate the uniform density of glycoproteins, specific for Con A binding, in the plasma membrane of normal and Gross virus leukemic mouse cells of different sizes. The utility of this procedure has also been demonstrated by using the mean fluorescence intensities of the distribution curves in the calculation of the fluorescence energy transfer efficiency.     

Quantification of cell-cycle distribution and mitotic index in Hydra by flow cytometry

The applicability of flow cytometry (FCM) to analyse cell-cycle distribution and mitotic cells in Hydra oligactis and Hydra vulgaris is demonstrated. The freshwater polyps H. vulgaris and H. oligactis are well-accepted animal models for studying cell proliferation, regeneration and differentiation. Disintegrated animals were labelled for FCM analysis according to the method of Nuesse et al. [(1990) Flow cytometric analysis of G(1) and G(2)/M-phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements. Cytometry 11, 813]. Proliferation and regeneration experiments, in the absence or presence of the oligopeptide head activator, were quantified. Cell-cycle analysis of different parts of the animals shows low proliferation in the head region and high proliferation in the gastric and foot regions. Cell-cycle analysis of different parts of Hydra, comparison of H. oligactis and H. vulgaris, as well as pharmacological treatment, yielded results that are in agreement with prior microscopic analysis. Our results demonstrate that FCM is an appropriate technique for quantifying proliferation in this animal model. It can be used for basic research on development, regeneration and differentiation as well as for innovative drug investigation and toxicology studies.     

正常人及不育者精子核DNA的定量研究

用流式细胞术,Feulgen显微分光光度法,荧光显微分光光度法测定正常人精子核DNA相对含量:结果表明,上述方法所测得的精子核DNA相对含量稳定,变化范围小。同时用Feulgen显微分光光度法测定不育者精子核DNA相对含量。显示不育者精子核DNA相对含量高于正常人,提示精子核DNA核蛋白复合物异常可能是某些男性不育症的原因。此项研究为诊断男性不育症提供了新方法,在男性学精子核分子研究方面提供了依据。     

Contribution of automated hematology analysis to the detection of apoptosis in peripheral blood lymphocytes

Automated hematology analyzers (analyzers) can provide complete blood counts and white blood cell (WBC) differentials in clinical laboratories and alert users to the presence of quantitative and qualitative cell abnormalities through cautionary flags. In this study, we applied analyzers to the screening of apoptotic cells in peripheral blood and examined the triggering capacity of cautionary flags to detect apoptotic cell populations. EDTA-anticoagulated fresh peripheral blood from patients with acute infectious mononucleosis containing atypical lymphocytes comprising 12.3 +/- 4. 0% of WBC was applied to a Beckman-Coulter MAXM A/L Retic (MAXM) analyzer. The lymphocyte cluster spread upward in VOLUME/DF1 scattergrams and the threshold lines between lymphocyte and monocyte clusters shifted upward. Flags for the number and percentage of lymphocytes, variant lymphocytes, and blast cells were generally present for samples containing atypical lymphocytes. After the blood from acute infectious mononucleosis patients was incubated for 4 h at 37 degrees C, peripheral blood smears revealed the presence of morphologically apoptotic cells comprising 9.0 +/- 4.2% of WBC and a comparable reduction of lymphocytes. On the MAXM analyzer, the apoptotic lymphocyte cluster appeared under the lymphocyte cluster in VOLUME/DF1 scattergrams. However, no specific flag was present to alert users to the presence of the apoptotic lymphocyte cluster. We conclude that visual inspection of scattergrams generated by the MAXM analyzer can be useful for the detection of apoptotic lymphocytes in peripheral blood. Cytometry (Comm. Clin. Cytometry) 42:209-214, 2000. Published 2000 Wiley-Liss, Inc.     

Rapid monitoring of cell size, vitality and lipid droplet development in the oleaginous yeast Waltomyces lipofer

The aim of this work was the development of rapid methods suitable for monitoring the growth of the oleaginous yeast Waltomyces lipofer by means of cell size, vitality and the development of internal lipid droplets throughout different growth phases. Oleaginous yeasts are of interest for the industrial production of lipids and therefore precise monitoring of growth characteristics is needed.This paper provides information about both the method development as well as about examples for their use in monitoring applications. Cell size and shape were determined using FPIA (Flow Particle Image Analysis). Vitality and internal lipid droplets were measured using two independent staining methods for Flow Cytometry. Double staining with cFDA & PI was used for the distinction between “vital”, “sublethal” and “dead” subpopulations, whereas Nile Red allowed the monitoring of lipid accumulation. In this approach the method for vitality measurement was optimized focussing on the staining buffer. An addition of 25 mM citric acid and pH 4.8 revealed to be optimal. The cells in the growth experiment showed a constantly high vitality, which was always above 90%, but slowly decreasing over time. In the course of lipid droplet development it could be seen that the cell size and the Nile Red fluorescence intensity increased. It was demonstrated that the tested method combination provides a powerful tool for rapid fermentation monitoring of the oleaginous yeast W. lipofer, which allows gaining information about the desired growth characteristics in less than 45 min. Further applications for the two methods will be discussed in this article.     

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuberculosis infection.     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

Improved model for specimen classification based on single-cell classifiers

We consider probabilistic models for specimen classification procedures based on systems which classify individual cells as normal or abnormal. The models which we consider generalize those discussed previously by Castleman and White (Anal. Quant. Cytol. 2:117-122, 1980; Cytometry 2:155-158, 1981) and by Timmers and Gelsema (Cytometry 6:22-25, 1985). In particular, they include the biologically plausible possibility that the specimen contains cells which are intermediate between the extremes of normal and abnormal. We find that if these additional cells occur differentially in normal and abnormal specimens, then specimen classification can become substantially more efficient when the cell classifier has different error rates for these cells.     

GenePattern flow cytometry suite

Background

Traditional flow cytometry data analysis is largely based on interactive and time consuming analysis of series two dimensional representations of up to 20 dimensional data. Recent technological advances have increased the amount of data generated by the technology and outpaced the development of data analysis approaches. While there are advanced tools available, including many R/BioConductor packages, these are only accessible programmatically and therefore out of reach for most experimentalists. GenePattern is a powerful genomic analysis platform with over 200 tools for analysis of gene expression, proteomics, and other data. A web-based interface provides easy access to these tools and allows the creation of automated analysis pipelines enabling reproducible research.

Results

In order to bring advanced flow cytometry data analysis tools to experimentalists without programmatic skills, we developed the GenePattern Flow Cytometry Suite. It contains 34 open source GenePattern flow cytometry modules covering methods from basic processing of flow cytometry standard (i.e., FCS) files to advanced algorithms for automated identification of cell populations, normalization and quality assessment. Internally, these modules leverage from functionality developed in R/BioConductor. Using the GenePattern web-based interface, they can be connected to build analytical pipelines.

Conclusions

GenePattern Flow Cytometry Suite brings advanced flow cytometry data analysis capabilities to users with minimal computer skills. Functionality previously available only to skilled bioinformaticians is now easily accessible from a web browser.

     

Estimating the Total Rate of DNA Replication Using Branching Processes

Increasing the knowledge of various cell cycle kinetic parameters, such as the length of the cell cycle and its different phases, is of considerable importance for several purposes including tumor diagnostics and treatment in clinical health care and a deepened understanding of tumor growth mechanisms. Of particular interest as a prognostic factor in different cancer forms is the S phase, during which DNA is replicated. In the present paper, we estimate the DNA replication rate and the S phase length from bromodeoxyuridine-DNA flow cytometry data. The mathematical analysis is based on a branching process model, paired with an assumed gamma distribution for the S phase duration, with which the DNA distribution of S phase cells can be expressed in terms of the DNA replication rate. Flow cytometry data typically contains rather large measurement variations, however, and we employ nonparametric deconvolution to estimate the underlying DNA distribution of S phase cells; an estimate of the DNA replication rate is then provided by this distribution and the mathematical model.