bio-flow-cytometry-fcs-handling

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.

FCS File Handling

"Load my FCS files into R or Python" → Read Flow Cytometry Standard (FCS) files, access channel parameters and metadata, and explore event data for downstream analysis.

• R: flowCore::read.FCS() or flowCore::read.flowSet() for multiple files
• Python: fcsparser.parse() or FlowCal.io.FCSData()

Load FCS Files

Goal: Read a single FCS file and inspect its parameters and metadata.

Approach: Use flowCore's read.FCS with transformation disabled to load raw data, then examine parameter names and descriptions.

library(flowCore)

# Read single FCS file
fcs <- read.FCS('sample.fcs', transformation = FALSE, truncate_max_range = FALSE)

# File info
print(fcs)

# Parameter names
colnames(fcs)  # Short names
pData(parameters(fcs))  # Full metadata including descriptions

Load Multiple Files

Goal: Read a batch of FCS files into a single flowSet container for uniform processing.

Approach: List FCS files from a directory and load them into a flowSet with read.flowSet.

# Read multiple files into flowSet
files <- list.files('data', pattern = '\\.fcs$', full.names = TRUE)
fs <- read.flowSet(files, transformation = FALSE, truncate_max_range = FALSE)

# Sample names
sampleNames(fs)

# Access individual samples
fcs1 <- fs[[1]]

Access Expression Data

Goal: Extract the expression matrix from a flowFrame for numeric analysis.

Approach: Call exprs() to get the cells-by-channels matrix, then subset or summarize as needed.

# Get expression matrix
expr <- exprs(fcs)
head(expr)

# Dimensions
dim(expr)  # cells x channels

# Channel statistics
summary(expr)

# Get specific channels
cd4_expr <- expr[, 'CD4']

Channel Metadata

Goal: Retrieve channel names, descriptions, and ranges from the FCS parameter table.

Approach: Access the parameters slot via pData(parameters(fcs)) and build a short-name to description mapping.

# Parameter information
params <- pData(parameters(fcs))
print(params)

# Parameter columns:
# - name: short name (e.g., "FL1-A")
# - desc: description (e.g., "CD4")
# - range: max value
# - minRange: min value

# Get channel mapping
channel_map <- setNames(params$desc, params$name)

Rename Channels

# Rename using descriptions
rename_channels <- function(fcs) {
    params <- pData(parameters(fcs))
    new_names <- ifelse(is.na(params$desc) | params$desc == '',
                        params$name, params$desc)
    colnames(fcs) <- new_names
    return(fcs)
}

fcs_renamed <- rename_channels(fcs)

Subsetting Data

# Subset by cells (rows)
fcs_subset <- fcs[1:1000, ]

# Subset by channels (columns)
fcs_markers <- fcs[, c('CD4', 'CD8', 'CD3')]

# Subset by expression values
high_cd4 <- fcs[exprs(fcs)[, 'CD4'] > 1000, ]

Merge flowSets

# Combine multiple flowSets
fs_combined <- rbind2(fs1, fs2)

# Or concatenate into single flowFrame
all_data <- fsApply(fs, exprs)
all_data <- do.call(rbind, all_data)

Write FCS Files

# Write single file
write.FCS(fcs, 'output.fcs')

# Write flowSet
write.flowSet(fs, outdir = 'output_dir')

Sample Metadata

# Add sample annotations
pData(fs) <- data.frame(
    name = sampleNames(fs),
    condition = c('Control', 'Control', 'Treatment', 'Treatment'),
    patient = c('P1', 'P2', 'P1', 'P2')
)

# Access
pData(fs)

Basic Visualization

library(ggcyto)

# Density plot
autoplot(fcs, 'FSC-A')

# Bivariate plot
autoplot(fcs, 'CD4', 'CD8')

# Multiple samples
autoplot(fs, 'CD4', 'CD8')

Check Data Quality

# Time parameter check
if ('Time' %in% colnames(fcs)) {
    time <- exprs(fcs)[, 'Time']
    plot(time, type = 'l', main = 'Acquisition Time')
}

# Event count per file
fsApply(fs, nrow)

# Check for saturated events
saturation <- apply(exprs(fcs), 2, function(x) mean(x == max(x)) * 100)
print(saturation)

Convert to Data Frame

# For use with tidyverse
library(tidyverse)

df <- as.data.frame(exprs(fcs))
df$sample <- 'sample1'

# From flowSet
df_all <- fsApply(fs, function(f) {
    d <- as.data.frame(exprs(f))
    d$sample <- identifier(f)
    d
}, simplify = FALSE)
df_all <- bind_rows(df_all)

Related Skills

• compensation-transformation - Apply compensation and transforms
• gating-analysis - Define cell populations
• clustering-phenotyping - Unsupervised analysis