Version Compatibility

Reference examples tested with: flowCore 2.14+, scanpy 1.10+

Before using code patterns, verify installed versions match. If versions differ:

• R: packageVersion('<pkg>') then ?function_name to verify parameters

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Compensation and Transformation

"Compensate and transform my flow cytometry data" → Correct spectral overlap between fluorophores using a compensation matrix and apply biexponential/arcsinh transforms for visualization and analysis.

• R: flowCore::compensate() then flowCore::transform() with estimateLogicle()

Load Compensation Matrix

library(flowCore)

# From FCS file keywords
fcs <- read.FCS('sample.fcs', transformation = FALSE)
comp_matrix <- keyword(fcs)$`$SPILLOVER`

# Or from CSV file
comp_matrix <- as.matrix(read.csv('compensation.csv', row.names = 1))

Apply Compensation

# Create compensation object
comp <- compensation(comp_matrix)

# Apply to flowFrame
fcs_comp <- compensate(fcs, comp)

# Apply to flowSet
fs_comp <- compensate(fs, comp)

Calculate Compensation from Controls

library(flowStats)

# Single-stained controls
controls <- read.flowSet(list.files('controls', pattern = '\\.fcs$', full.names = TRUE))

# Calculate spillover matrix
spillover <- spillover(controls,
                        unstained = 'Unstained.fcs',
                        fsc = 'FSC-A', ssc = 'SSC-A',
                        patt = '-A$',  # Channel pattern
                        stain_match = 'regexpr')

# The result is a list; extract matrix
comp_matrix <- spillover$comp