SPEEDINESS OVERLAY SOFTWARE
Flow is a software application and interface to expose research and clinical cytometrists to these powerful techniques that will surely become more important as the complexity and dimensionality of flow technology increases. There has been increasing interest in the potential utility of statistical clustering and specifically the use of mixture models for flow cytometry, as shown by recent publications in Cytometry A, the official journal of the International Society for Analytical Cytology (ISAC). As Flow is both open source and extensible by design with third-party plugins, we encourage the user community to provide additional functionality, and provide a developer's guide to the API on our website. While the current features of Flow are naturally biased toward our own research interests, we have implemented features that allow the software to be used for traditional analysis by gating. Together, these provide powerful new tools for the experimentalist or clinician to analyze flow cytometric data in a different way – one that will complement and perhaps supersede the traditional strategy of sequential gating. Nevertheless, we believe that statistical modeling can ameliorate much of the tedium and subjectivity inherent in gating on PFC data, and that Flow is a useful medium to increase awareness of such methods among the flow community.įlow is an open source software application for flow cytometric analysis that integrates tools for exploratory data analysis, clustering and annotation of flow cytometric data sets. In practice, accurate gating is often only possible with extensive expert knowledge and by comparison with both negative and positive controls, and formidable statistical challenges exist. The motivating idea is that if a subset of cells forms a distinct visual subgroup that can be demarcated visually with sequential gating, it should also be possible in principle to extract the subset with the appropriate statistical model. In addition to the standard histogram and dot plot views of flow data, Flow also provides graphics to visualize data in three dimensions, along with the ability to rotate, pan and zoom into or out of the 3D view. The analysis of PFC is largely a process of trying to find structure in a high-dimensional space, and visualization is an invaluable aid to such exploratory data analysis. Since the reliable identification of cell subsets is necessary for further investigation of their function, this problem affects downstream research as well. The lack of software suitable for experimentalists and clinicians to efficiently and robustly characterize flow data is a bottleneck in the use of PFC.
SPEEDINESS OVERLAY MANUAL
The main problem with manual gating is that it is highly operator-dependent and subjective, with the choice of gating sequence and position chosen by expertise, making replication of results by a different laboratory extremely difficult. With today's software, flow cytometric datasets are typically analyzed by manually circumscribing (gating) interesting regions using a sequence of 1 or 2-dimensional plots to identify different subsets. Polychromatic flow cytometry (PFC) is the only currently available assay that can track multiple functional responses simultaneously, on both single cell and population levels, and uses include immunophenotyping, monitoring of complex immune responses in HIV patients and vaccine trials and characterization of cell signaling with labeled antibodies to phosphoproteins.
With recent technological advances, a single session may collect high-resolution digital events from up to a million cells, each labeled with up to 20 different fluorochromes, resulting in a data set that is both high volume and multi-dimensional.
Since each fluorochrome emits light of a specific color when activated, the density of tagged molecules on each cell can be estimated. In flow cytometry, intracellular or cell surface molecules (markers) in a cell population are tagged with one or more fluorescent dyes, then streamed down a flow cell where the fluorochromes are activated by multiple lasers. Flow cytometry is a high throughput method extensively used in both experimental and clinical settings for characterizing cell phenotypes, especially in the disciplines of hematology, immunology and infectious diseases.
0 kommentar(er)
