bio-flow-cytometry-differential-analysis

Version Compatibility

Reference examples tested with: R stats (base), edgeR 4.0+, ggplot2 3.5+, limma 3.58+

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.

Differential Analysis

"Compare cell populations between my conditions" → Test for significant changes in cell type frequencies (differential abundance) or marker expression levels (differential state) between experimental groups.

• R: CATALYST::testDA_edgeR() or diffcyt::testDA_GLMM()

Differential Abundance (DA)

Goal: Test which cell population clusters differ in frequency between experimental conditions.

Approach: Create a design matrix and contrast from sample metadata, then run edgeR-based differential abundance testing on cluster counts per sample using testDA_edgeR from the diffcyt framework.

library(CATALYST)
library(diffcyt)

# Load clustered data
sce <- readRDS('sce_clustered.rds')

# Create design matrix
design <- createDesignMatrix(ei(sce), cols_design = 'condition')

# Create contrast
contrast <- createContrast(c(0, 1))  # Treatment vs Control

# Differential abundance test
res_DA <- testDA_edgeR(sce, design, contrast, cluster_id = 'meta20')

# View results
rowData(res_DA)$cluster_id
rowData(res_DA)$p_adj

# Significant clusters
sig_DA <- rowData(res_DA)$p_adj < 0.05
table(sig_DA)

Differential State (DS)

# Test for marker expression differences within clusters
res_DS <- testDS_limma(sce, design, contrast,
                        cluster_id = 'meta20',
                        markers_include = rownames(sce)[rowData(sce)$marker_class == 'state'])

# Results per marker per cluster
ds_results <- rowData(res_DS)

Visualization

# DA results heatmap
plotDiffHeatmap(sce, res_DA, all = TRUE, fdr = 0.05)

# DS results heatmap
plotDiffHeatmap(sce, res_DS, all = TRUE, fdr = 0.05)

# Abundance by condition
plotAbundances(sce, k = 'meta20', by = 'cluster_id', group_by = 'condition')

Manual Statistical Testing

library(tidyverse)

# Get cluster frequencies per sample
freqs <- colData(sce) %>%
    as.data.frame() %>%
    group_by(sample_id, condition, cluster_id = cluster_ids(sce, 'meta20')) %>%
    summarise(n = n(), .groups = 'drop') %>%
    group_by(sample_id) %>%
    mutate(freq = n / sum(n) * 100)

# Test each cluster
test_abundance <- function(df, cluster) {
    cluster_data <- filter(df, cluster_id == cluster)
    ctrl <- filter(cluster_data, condition == 'Control')$freq
    treat <- filter(cluster_data, condition == 'Treatment')$freq

    if (length(ctrl) >= 2 && length(treat) >= 2) {
        test <- t.test(treat, ctrl)
        return(data.frame(
            cluster = cluster,
            fc = mean(treat) / mean(ctrl),
            pvalue = test$p.value
        ))
    }
    return(NULL)
}

results <- map_dfr(unique(freqs$cluster_id), ~test_abundance(freqs, .x))
results$padj <- p.adjust(results$pvalue, method = 'BH')

Mixed Effects Models

library(lme4)
library(lmerTest)

# For paired/repeated measures designs
# Random effect for patient/donor

fit_mixed <- function(df, cluster) {
    cluster_data <- filter(df, cluster_id == cluster)

    model <- lmer(freq ~ condition + (1|patient_id), data = cluster_data)

    coef <- summary(model)$coefficients
    return(data.frame(
        cluster = cluster,
        estimate = coef[2, 'Estimate'],
        pvalue = coef[2, 'Pr(>|t|)']
    ))
}

CITRUS (Automated Discovery)

library(citrus)

# Prepare data
fcs_files <- list.files('data', pattern = '\\.fcs$', full.names = TRUE)
labels <- c(rep('Control', 2), rep('Treatment', 2))

# Run CITRUS
citrus_result <- citrus(
    fcs_files,
    labels,
    fileSampleSize = 1000,
    featureType = 'abundances',
    modelType = 'glmnet',
    family = 'classification'
)

# Get significant clusters
citrus_plot(citrus_result)

Volcano Plot

library(ggplot2)

# From DA results
da_df <- as.data.frame(rowData(res_DA))
da_df$significant <- da_df$p_adj < 0.05

ggplot(da_df, aes(x = logFC, y = -log10(p_adj), color = significant)) +
    geom_point() +
    geom_hline(yintercept = -log10(0.05), linetype = 'dashed') +
    geom_vline(xintercept = c(-1, 1), linetype = 'dashed') +
    scale_color_manual(values = c('gray', 'red')) +
    theme_bw() +
    labs(title = 'Differential Abundance')

Export Results

# Combine DA and DS results
da_results <- as.data.frame(rowData(res_DA))
da_results$analysis <- 'DA'

ds_results <- as.data.frame(rowData(res_DS))
ds_results$analysis <- 'DS'

# Save
write.csv(da_results, 'da_results.csv', row.names = FALSE)
write.csv(ds_results, 'ds_results.csv', row.names = FALSE)

Multiple Comparisons

# For multiple conditions
design_full <- model.matrix(~ 0 + condition, data = ei(sce))
colnames(design_full) <- levels(factor(ei(sce)$condition))

# Multiple contrasts
contrasts <- makeContrasts(
    TreatA_vs_Ctrl = TreatmentA - Control,
    TreatB_vs_Ctrl = TreatmentB - Control,
    TreatA_vs_B = TreatmentA - TreatmentB,
    levels = design_full
)

# Test each contrast
res_list <- lapply(1:ncol(contrasts), function(i) {
    testDA_edgeR(sce, design_full, contrasts[, i], cluster_id = 'meta20')
})

Related Skills

• clustering-phenotyping - Cluster data first
• gating-analysis - Compare gated populations
• differential-expression/de-results - Similar statistical concepts