spillover_ng-flowSet {flowStats} | R Documentation

## Compute a spillover matrix from a `flowSet`, simplified API

### 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`.
Matching stain channels to compensation controls is done via a matching csv file (at the path given by `matchfile`) with columns 'filename' and 'channel'. The 'channel' entries should exactly match the channel names in the FCS files. The 'filename' should be the FCS file name of each compensation control which should also be the corresponding sample name in the `flowSet`. There should also be one unstained control with the 'channel' entry of 'unstained'.
The method also allows for `x` to be missing if `path` is provided, pointing to a directory containing the control FCS files.
By default, pregating is always done on the channels using this API, and the mode of the channel is used to compute the spillover matrix. FSC and SSC channels can be provided to allow a pregating on (approximately) a population in the FSC and SSC dimensions. Also by default, a `norm2Filter` is applied before computing the spillover. These defaults can be overridden using the `pregate`, `method`, and `useNormFilt` arguments.

### Usage

    ## S4 method for signature 'flowSet'
    spillover_ng(x, fsc = "FSC-A", ssc = "SSC-A",
                 plot = FALSE, matchfile, path,
                 useNormFilt = TRUE, patt = NULL, pregate = TRUE, method = "mode", ...)
    ## S4 method for signature 'missing'
    spillover_ng(x, fsc = "FSC-A", ssc = "SSC-A",
                 plot = FALSE, matchfile, path,
                 useNormFilt = TRUE, patt = NULL, pregate = TRUE, method = "mode", ...)

    ## S4 method for signature 'missing'
    spillover_ng(x, fsc = "FSC-A", ssc = "SSC-A",
      plot = FALSE, matchfile, path, useNormFilt = TRUE, patt = NULL,
      pregate = TRUE, method = "mode", ...)

### Arguments

`x` | A flowSet of compensation beads or cells
`fsc` | The name or index of the forward scatter parameter
`ssc` | The name or index of the side scatter parameter
`plot` | logical. Plots the kernel density for each channel when pregating. Displays the gate used. If `pregate` is set to `FALSE`, this argument is ignored.
`matchfile` | Name of the csv file holding the compensation control file to channel matching information.
`path` | A path to a directory containing the control files, to be used if `x` is not provided.
`useNormFilt` | logical Indicating whether to apply a `norm2Filter` first before computing the spillover
`patt` | An optional regular expression defining which parameters should be considered
`pregate` | logical Indicating whether to pregate using `link{rangeGate}` before computing the spillover
`method` | The statistic to use for calculation. Traditionally, this has been the median so it is the default. The mean is sometimes more stable.
`...` | Additional arguments passed to `rangeGate`.

### 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

### 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`

[Package flowStats version 3.44.0 Index]