spillover-flowSet: Compute a spillover matrix from a flowSet

In RGLab/flowStats: Statistical methods for the analysis of flow cytometry data

Compute a spillover matrix from a flowSet

Description

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 names of each of the compensation control flowFrames (that is, sampleNames(x), which will typically be the filenames passed to read.FCS). By default, we must "guess" based on the largest statistic for the compensation control (i.e., the row).

Additionally, matching of channels to compensation control files can be accomplished using the spillover_match method, which allows the matches to be specified using a csv file. The flowSet returned by the spillover_match method should then be used as the x argument to spillover with prematched = TRUE.

Usage

## S4 method for signature 'flowSet'
spillover(
  x,
  unstained = NULL,
  fsc = "FSC-A",
  ssc = "SSC-A",
  patt = NULL,
  method = "median",
  stain_match = c("intensity", "ordered", "regexpr"),
  useNormFilt = FALSE,
  prematched = FALSE,
  exact_match = FALSE
)

Arguments

x	A flowSet of compensation beads or cells
unstained	The name or index of the unstained negative control
fsc	The name or index of the forward scatter parameter
ssc	The name or index of the side scatter parameter
patt	An optional regular expression defining which parameters should be considered
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 norm2Filter first before computing the spillover
prematched	a convenience argument specifying if the channels have already been matched by spillover_match. This will override the values of unstained and stain_match with unstained = "unstained" and stain_match = "regexpr".
exact_match	a logical specifying if we should use "regex" or "exact match" to match column names. The spillover_ng will pass exact_match and "regexpr" method will be over-ridden.

Details

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.

Value

A matrix for each of the parameters

Author(s)

B. Ellis, J. Wagner

References

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.

See Also

compensate, spillover_match

RGLab/flowStats documentation built on July 20, 2023, 1:33 a.m.