Clean doublet events from flow cytometry data

will adjust a polygon gate aimed at cleaning doublet events from the flowFrame. The main idea is to use the ratio between the two indicated channel as an indicator and select only the events for which this ratio is 'not too far' from the median ratio. More specifically, the computed ratio is ch1/(1+ch2). However, instead of looking at a constant range of this ratio, as is done in PeacoQC::removeDoublets(), which leads to a semi-conic gate, we apply a parallelogram shaped gate, by keeping a constant range of channel 2 intensity, based on the target ratio range at the mid value of channel 1.

singletsGate(
  ff,
  filterId = "Singlets",
  channel1 = "FSC-A",
  channel2 = "FSC-H",
  nmad = 4,
  verbose = FALSE
)

Arguments

ff

A flowCore::flowframe that contains flow cytometry data.

filterId

the name for the filter that is returned

channel1

The first channel that will be used to determine the doublet events. Default is "FSC-A"

channel2

The second channels that will be used to determine the doublet events. Default is "FSC-H"

nmad

Bandwidth above the ratio allowed (cells are kept if their ratio is smaller than the median ratio + nmad times the median absolute deviation of the ratios). Default is 4.

verbose

If set to TRUE, the median ratio and width will be printed. Default is FALSE.

Value

This function returns a flowCore::polygonGate.

Examples

data(OMIP021Samples)

# simple example with one single singlets gate filter 
# FSC-A and FSC-H channels are used by default

mySingletsGate <- singletsGate(OMIP021Samples[[1]], nmad = 3)

selectedSinglets <- flowCore::filter(
    OMIP021Samples[[1]],
    mySingletsGate)

ff_l <- flowCore::Subset(OMIP021Samples[[1]], selectedSinglets)

linRange <- c(0, 250000)

ggplotFilterEvents(
    ffPre = OMIP021Samples[[1]],
    ffPost = ff_l,
    xChannel = "FSC-A", xLinearRange = linRange,
    yChannel = "FSC-H", yLinearRange = linRange)


# application of two singlets gates one after the other

singletsGate1 <- singletsGate(OMIP021Samples[[1]], nmad = 3)
singletsGate2 <- singletsGate(OMIP021Samples[[1]],
                              channel1 = "SSC-A",
                              channel2 = "SSC-H",
                              filterId = "Singlets2")

singletCombinedGate <- singletsGate1 & singletsGate2

selectedSinglets <- flowCore::filter(
    OMIP021Samples[[1]],
    singletCombinedGate)

ff_l <- flowCore::Subset(OMIP021Samples[[1]], selectedSinglets)

ggplotFilterEvents(
    ffPre = OMIP021Samples[[1]],
    ffPost = ff_l,
    xChannel = "FSC-A", xLinearRange = linRange,
    yChannel = "FSC-H", yLinearRange = linRange)


ggplotFilterEvents(
    ffPre = OMIP021Samples[[1]],
    ffPost = ff_l,
    xChannel = "SSC-A", xLinearRange = linRange,
    yChannel = "SSC-H", yLinearRange = linRange)