%\VignetteIndexEntry{GateFinder}

\documentclass{article}

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\title{Projection-based Gating Strategy Optimization for Flow and Mass Cytometry}
\author{Nima Aghaeepour and Erin F. Simonds}

\begin{document}
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\setkeys{Gin}{width=1.0\textwidth, height=1.1\textwidth}

\maketitle
\begin{center}
{\tt naghaeep@gmail.com and erin.simonds@gmail.com}
\end{center}

\textnormal{\normalfont}

\tableofcontents

\section{Licensing}

Under the Artistic License, you are free to use and redistribute this software. 



\section{Introduction}
Exploratory analysis using polychromatic
\cite{chattopadhyay2006quantum} and mass \cite{bendall2011single} flow cytometry together with modern
computational tools (\emph{e.g.,}
\cite{aghaeepour2012early,aghaeepour2012rchyoptimyx,
  qiu2011extracting, amir2013visne, aghaeepour2013critical}) often result in identification of complex cell
populations that cannot be easily described using a limited number of
markers. GateFinder attempts to identify a series of gates (\emph{i.e.}
polygon filters on 2-dimensional scatter plots) that can discriminate
between a target cell population and other cells. 

Briefly, the analysis consists of three steps:
\begin{enumerate}
\item Project the data points into all possible pairs of
  dimensions. Use robust statistics to exclude outliers
  \cite{filzmoser2008outlier}. Calculate a convex hull (a convex
  polygon around the remaining data points) \cite{eddy1977new}.
  \item Calculate F-measure values for all available gates. Select the
    best one. 
    \item Depending on software configurations (see the $update.gates$
      parameter) either go to 1 or 2 unless the maximum number of iterations has
    been reached.
\end{enumerate}



\section{Basic Functionality}
This example uses part of a publicly available bone marrow mass cytometry dataset
\cite{bendall2011single}. In this specific subset of the dataset cells
were stimulated by lipopolysaccharide (LPS) and the response was
measured phosphorylation of p38 mitogen-activated protein kinase
(p38 MAPK). A random subset of $1000$ cells were selected for this
analysis to comply with BioConductor's size and run time
requirements. The optimal number of cells for GateFinder depends on the
number of parameters in the search space, the size of the target population,
and the desired purity. GateFinder expects transformed data. This dataset was
previously transformed with the \emph{arcsinhTransform()} function from the flowCore package, using
parameters $a=0$, $b=0.2$, $c=0$. Original analysis of the data revealed that the majority
of the p38 MAPK response is in the CD11b+ monocytes. Here, we will use GateFinder
to derive a specific gating strategy for the LPS-responsive cell population.

First, we select the target cell population by gating the phospho-p38 marker
(dimension number $34$) and selecting all cells with intensity greater than 3.5:
<<c1, echo=TRUE, fig=TRUE>>=
library(GateFinder)
library(flowCore)

data(LPSData)
targetpop <- (exprs(rawdata)[,34] > 3.5)
plot(exprs(rawdata)[ , c(2,34)], pch='.', col=targetpop+1, 
     xlab='Cell Length', ylab='p-p38')
abline(h=3.5, col=3, lwd=2, lty=2)
@ 

Next, we select the markers that should be considered for the gating
strategy and run the core \emph{GateFinder()} function:
<<c12, echo=TRUE, fig=FALSE>>=
x=exprs(rawdata)[ , prop.markers]
colnames(x)=marker.names[prop.markers]
results=GateFinder(x, targetpop)
@

Now we can create a scatter plot of each gating step. GateFinder's
\emph{plot.GateFinder()} function accepts 4 arguments specifying the raw data, the
output of the \emph{GateFinder()} function, the layout of figure panels to
assemble in the plot, and a logical mask specifying the target cells.
The original target cells are highlighted in red. Gray cells were excluded
in one of the previous gating steps. Black cells are cells that are not
in the original target population. This analysis suggests that the
target population is CD33$^+$CD34$^+$CD11b$^+$CD3$^+$.
<<c13, echo=TRUE, fig=TRUE>>=
plot (x, results, c(2,3), targetpop)
@

We can also visualize the F-measure, precision (i.e., ``purity''), 
and recall (i.e., ``yield'') of each
step. As expected, making the gating more strict (by including more
gating steps) increases the precision and decreases the recall of the
gating strategy.
<<c14, echo=TRUE, fig=TRUE>>=
plot(results)
@ 


\section{Advanced Parameters}
GateFinder's functionality can be controled using two parameters: the
\emph{outlier.percentile} value controls the robustness of the convex
hulls (polygon gates) to outliers and the \emph{beta} value controls
the relative impact of precision and recall on the F-measure calculations.


Higher values for the \emph{outlier.percentile} parameter make the
gates less strict (and therefore will increase precision and decrease recall):
<<c2, echo=TRUE, fig=TRUE>>=
results=GateFinder(x, targetpop, outlier.percentile=0.5)     
plot (x, results, c(2,3), targetpop)
plot(results)
@ 

Similarly, a \emph{beta} value smaller than $1$ increases in the impact
of precision on the F-measure calculations. In the following
calculations a value of 0.5 makes precision twice as important as
recall. Therefore the algorithm modifies the gating strategy to
increase the precision of the gating strategy. This is achieved by
combining CD11b and CD33 in the very first gate at the cost of a
decreased recall.
<<c21, echo=TRUE, fig=TRUE>>=
results=GateFinder(x, targetpop, beta=0.5)     
plot (x, results, c(2,3), targetpop)
plot(results)
@ 

\bibliographystyle{plain}
\bibliography{GateFinder}


\end{document}
