spillover {flowCore} | R Documentation |
Spillover information for a particular experiment is often
obtained by running several tubes of beads or cells stained with
a single color that can then be used to determine a spillover
matrix for use with compensate
.
When matching stain channels in x
with the compensation controls, we
provide a few options. If ordered
, we assume the ordering of the channels
in the flowSet object is the same as the ordering of the compensation-control
samples. IF regexpr
, we use a regular expression to match the channel
names with the filenames of the compensation controls. By default, we must
"guess" based on the largest statistic for the compensation control (i.e., the
row).
## S4 method for signature 'flowSet' spillover(x, unstained = NULL, patt = NULL, fsc = "FSC-A", ssc = "SSC-A", method = "median", stain_match = c("intensity", "ordered", "regexpr"), useNormFilt=FALSE, pregate = FALSE, plot = FALSE, ...)
x |
A flowSet of compensation beads or cells |
unstained |
The name of index of the unstained negative control |
patt |
An optional regular expression defining which parameters should be considered. |
fsc |
The name or index of the forward scatter parameter |
ssc |
The name or index of the side scatter parameter |
method |
The statistic to use for calculation. Traditionally, this has been the median so it is the default. The mean is sometimes more stable. |
stain_match |
Determines how the stain channels are matched with the compensation controls. See details. |
useNormFilt |
Logical Indicating whether to apply a
|
pregate |
logical. Should we pregate each channel before computing the spillover matrix? By default, no. |
plot |
logical. Plots the kernel density for each channel when
pregating. Displays the gate used. If |
... |
Additional arguments passed to |
The algorithm used is fairly simple. First, using the scatter parameters, we restrict ourselves to the most closely clustered population to reduce the amount of debris. The selected statistic is then calculated on all appropriate parameters and the unstained values swept out of the matrix. Every sample is then normalized to [0,1] with respect to the maximum value of the sample, giving the spillover in terms of a proportion of the primary channel intensity.
A matrix for each of the parameters
B. Ellis
C. B. Bagwell \& E. G. Adams (1993). Fluorescence spectral overlap compensation for any number of flow cytometry parameters. in: Annals of the New York Academy of Sciences, 677:167-184.