Compute Spillover Matrix - GatingSet 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 GatingSet
spillover_compute(x, parent = NULL,
  axes_trans = NULL, channel_match = NULL,
  spillover = "Spillover-Matrix.csv", ...)

Arguments

x	object of class GatingSet 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 and indicate the population of interest using the parent argument.
parent	name of the pre-gated population to use for downstream calculations, set to the last node of the GatingSet by default (e.g. "Single Cells").
axes_trans	object of class transformerList 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 GatingSet containing gated single stain compensation controls and an unstained control. spillover_compute uses the method described by Bagwell & Adams 1993 to calculate fluorescent spillover values using single stain compensation controls and a universal 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(gs,
  parent = "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)