Biomedical Engineering Reference
In-Depth Information
genes. The phosphorylation of STATs is mediated mainly by Janus kinases (JAKs).
The JAK/STAT pathway plays a critical role in hematopoietic and immune cell
function. The assay formats available to detect, for example, phosphorylated STAT5
(pSTAT5) have relatively low throughput and involve lengthy protocols including
those for flow cytometry. Recently, an alternative homogeneous assay has been
described. The SureFire (Perkin Elmer, Waltham, MA) pSTAT5 assay utilizes
AlphaScreen (Perkin Elmer) technology to detect pSTAT5 in cell lysates, amenable
to high-throughput screening applications [150].
2.6 FACING THE CHALLENGE OF HIGH-THROUGHPUT
FLOW CYTOMETRY DATA ANALYSIS
Data analysis techniques in the larger flow community remain embryonic and
represent the major area in flow cytometry from where new developments will
emerge over the forthcoming decade [151]. Typically, current high-throughput
methods can generate up to a thousand flow cytometry data files per day and each
data file can consist of millions of multiparametric descriptions of individual cells.
A simple time series experiment for high-throughput analysis encompasses a screen-
ing matrix consisting of typically nine cell types and a treatment regimen requiring 16
treatments with corresponding baseline (controls) and usually sampled at different
stimulation time points; one single treatment regimen contains more than 12 million
data points. This highlights the principal data challenge facing the field: automated
cluster analysis of high-content flow cytometry data.
2.6.1 Manual Analysis of Data Presents Limitations
Manual data analysis (using software packages such as FlowJo (Tree Star Inc.) and
FCSExpress (DeNovo Software)) is typically undertaken and appropriate for hypoth-
esis-driven interrogation to extract quantitative distributions and parameter relation-
ships, but is essentially limited by the capacity of the researcher to only visualize and
interrogate three-dimensional data. These packages enhance user capacity to organize
and group data as well as provide tools to segment (gate) subpopulations in histograms
and also cascade appropriate gating using Boolean expressions to different data sets.
Manual analysis is, however, not suitable for tackling a drug screen or multicolor
clinical investigations; any process that requires manual intervention often results in
issues of subjectivity, generation of errors, and low throughput providing a series of
hurdles to reproducible data analysis.
2.6.2 Systematic Methodologies for Data Analysis
High-throughput data demand systematic computational approaches to analyze and
rationally interpret data screens. Open source packages such as Bioconductor for
analysis of flow cytometry data provide an integrated framework for bioinformati-
cians, statisticians, and computational biologists to develop methods to analyze and
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