Compute Spillover Matrix - flowSet Method

spillover_compute uses the method described by Bagwell & Adams 1993 to calculate the fluorescent spillover matrix using a reference universal unstained control and single stain compensation controls.

# S4 method for flowSet
spillover_compute(x, axes_trans = NULL,
  channel_match = NULL, spillover = "Spillover-Matrix.csv", ...)

Arguments

x	object of class flowSet containing pre-gated single stain compensation controls and a universal unstained control. Currently, spillover_compute does not pre-gate samples to obtain a homogeneous cell population for downstream calculations. We therefore recommend pre-gating samples based on FSC and SSC parameters prior to passing them to spillover_compute (i.e. x should contain events for single cells only). Passing raw files to spillover_compute will result in inaccurate calculations of fluorescent spillover matrix.
axes_trans	object of class transformList generated by estimateLogicle to transform fluorescent channels for gating. axes_trans is required if logicle transformation has already been applied to x using estimateLogicle. spillover_compute will automatically call estimateLogicle internally to transform channels prior to gating, if axes_trans is supplied it will be used for the transformation instead.
channel_match	name of .csv file containing the names of the samples in a column called "name" and their matching channel in a column called "channel". spillover_compute will the guide you through the channel selection process and generate a channel match file called "Compensation-Channels.csv" automatically. If you already have a complete channel_match and would like to bypass the channel selection process, simply pass the name of the channel_match to this argument (e.g. "Compensation-Channels.csv").
spillover	name of the output spillover csv file, set to "Spillover-Matrix.csv" by default.
...	additional arguments passed to cyto_plot,flowFrame-method.

Value

spillover matrix object and "Spillover Matrix.csv" file.

Details

Calculate spillover matrix using flowSet containing gated single stain compensation controls and an unstained control. spillover_compute begins by the user selecting which fluorescent channel is associated with each control from a dropdown menu. Following channel selection, spillover_compute runs through each control and plots the density distribution of the unstained control in red and the compensation control in blue. Users can then gate the positive signal for spillover calculation using an interval gate. The percentage spillover is calculated based on the median fluorescent intensities of the stained populations and the universal unstained sample. The computed spillover matrix is returned as an R object and written to a named .csv file for future use.

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

cyto_plot,flowFrame-method

Examples

library(CytoRSuiteData)

# Bypass directory check for external files
options("CytoRSuite_wd_check" = FALSE)

# Don't run - skips the gating process
options("CytoRSuite_interact" = FALSE)

# Load in compensation controls
fs <- Compensation
gs <- GatingSet(Compensation)
#> .
#> .
#> .
#> .
#> .
#> .
#> done!

# Gate using gate_draw
gt <- Compensation_gatingTemplate
gating(gt, gs)
#> Preprocessing for 'Cells'
#> Gating for 'Cells'
#> done.
#> Preprocessing for 'Single Cells'
#> Gating for 'Single Cells'
#> done.
#> finished.

# Channel match fille
cmfile <- system.file("extdata",
  "Compensation-Channels.csv",
  package = "CytoRSuiteData"
)

# Compute fluorescent spillover matrix
spill <- spillover_compute(getData(gs, "Single Cells"),
  channel_match = cmfile,
  spillover = "Example-spillover.csv"
)

# Compensate samples
gs <- compensate(gs, spill)

# Return CytoRSuite_wd_check to default
options("CytoRSuite_wd_check" = TRUE)

# Return CytoRSuite_interact to default
options("CytoRSuite_interact" = TRUE)