Version Compatibility

Reference examples tested with: CATALYST 1.26+, diffcyt 1.22+, FlowSOM 2.10+, flowCore 2.14+, flowWorkspace 4.14+, flowStats 4.14+, edgeR 4.0+, limma 3.58+, ggplot2 3.5+; Python (partial alt) flowkit 1.1+.

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

• R: packageVersion('<pkg>') then ?function_name to verify parameters
• Python: pip show <package> then help(module.function) to check signatures

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt rather than retrying. Each stage defers depth to its component skill.

Flow Cytometry Pipeline

"Process my cytometry data from FCS to differential populations" -> read raw -> compensate/unmix -> transform -> QC -> remove doublets -> cluster (or gate) -> annotate -> test DA/DS, with the sample as the unit of inference.

• R: flowCore + CATALYST::prepData/cluster/runDR + diffcyt::diffcyt()

The Single Most Important Modern Insight -- A Pipeline Is a Chain of Irreversible Decisions, and the Unit of Inference Is the Sample

Each early choice silently gates the validity of the final test: reading raw (not log-linearized), compensating BEFORE transforming, removing margin events before density QC, assigning type-vs-state markers correctly, and removing doublets before clustering. None of these is recoverable downstream - a doublet clustered as a "double-positive," a state marker used for clustering, or an uncompensated channel becomes a false population that the differential test then "confirms." The second critical thread is that the SAMPLE/subject, not the cell, is the experimental unit: diffcyt aggregates cells to per-sample-per-cluster counts (DA) and medians (DS) before testing, so biological replication (>= 2-3 per group) is mandatory and a per-cell test is invalid. Two normalization layers sit at different points in the pipeline - EQ-bead drift correction on raw counts at the very front (CyTOF), and CytoNorm cross-batch harmonization on transformed data before the analytical clustering (its internal FlowSOM clustering is part of the batch model, not the analysis) - and conflating them is a classic error.

Decision Tree: Which Path

Pipeline Overview

FCS -> compensate/unmix -> transform -> QC (margins, time, dead) -> doublets
     -> [ cluster (FlowSOM) | gate (openCyto) ] -> annotate -> diffcyt DA/DS -> report
EQ-bead drift normalization (CyTOF) runs on raw counts BEFORE everything; CytoNorm runs on transformed data and its normalized output feeds the cluster/gate step.

1. Panel, Metadata, and Load

Goal: Define the type/state panel and sample metadata, then load FCS.

Approach: Panel marker_class drives everything downstream (type clusters, state is tested); metadata keys samples to condition/subject. See flow-cytometry/fcs-handling.

library(CATALYST); library(diffcyt); library(flowCore); library(ggplot2)

panel <- data.frame(
  fcs_colname = c('FSC-A','SSC-A','CD45','CD3','CD4','CD8','CD19','CD14','Ki67','IFNg'),
  antigen     = c('FSC','SSC','CD45','CD3','CD4','CD8','CD19','CD14','Ki67','IFNg'),
  marker_class = c('none','none','type','type','type','type','type','type','state','state'))
md <- data.frame(file_name = list.files('data', pattern = '\\.fcs$'),
                 sample_id = paste0('S', 1:8),
                 condition = rep(c('Control','Treatment'), each = 4),
                 patient_id = rep(paste0('P', 1:4), 2))
fs <- read.flowSet(file.path('data', md$file_name), transformation = FALSE, truncate_max_range = FALSE)

2. Compensate / Unmix, then Transform

Goal: Remove spillover on linear data, then variance-stabilize.

Approach: Conventional flow compensates (matrix before transform); CyTOF skips fluorescence compensation and uses cofactor 5; spectral unmixes then uses ~150. See flow-cytometry/compensation-transformation.

spill <- spillover(fs[[1]]); spill <- spill[[which(!vapply(spill, is.null, logical(1)))[1]]]  # first POPULATED matrix; FACS stores it under SPILL/$SPILLOVER, not always [[1]]
fs_comp <- compensate(fs, spill)                        # conventional flow; CyTOF: omit or use compCytof
COFACTOR <- 150                                          # 5 for CyTOF, ~150 for fluorescence/spectral
sce <- prepData(fs_comp, panel, md, transform = TRUE, cofactor = COFACTOR, FACS = TRUE)

3. QC (order matters)

Goal: Remove margin/boundary events and time anomalies before any density step.

Approach: Margins first, then time-based cleaning; on CyTOF, EQ-bead drift correction happens upstream on raw counts. See flow-cytometry/cytometry-qc and flow-cytometry/bead-normalization.

# per-sample sanity + sample-similarity MDS (flag outlier samples)
plotExprs(sce, color_by = 'condition'); pbMDS(sce, color_by = 'condition')
# event-level cleaning runs per-FCS upstream: PeacoQC::RemoveMargins() -> PeacoQC()/flowAI on transformed data

4. Remove Doublets

Goal: Drop aggregates before clustering so they don't form phantom double-positives.

Approach: Flow uses the FSC-A vs FSC-H diagonal; CyTOF uses DNA intercalator + Gaussian/Event_length. See flow-cytometry/doublet-detection.

# CyTOF (FACS=TRUE retained Event_length on the arcsinh scale):
e <- assay(sce, 'exprs')
if (all(c('DNA1','Event_length') %in% rownames(sce))) {
  keep <- e['DNA1', ] > quantile(e['DNA1', ], 0.05) &
          e['Event_length', ] <= quantile(e['Event_length', ], 0.99)
  sce <- sce[, keep]
}

5. Cluster (FlowSOM) or Gate

Goal: Define populations by unsupervised clustering on TYPE markers (discovery) or hierarchical gating (defined/rare).

Approach: cluster() wraps FlowSOM+ConsensusClusterPlus; over-provision the grid, set a seed. See flow-cytometry/clustering-phenotyping (clustering) and flow-cytometry/gating-analysis (gating).

sce <- cluster(sce, features = 'type', xdim = 10, ydim = 10, maxK = 20, seed = 42)

6. Annotate and Visualize Structure

Goal: Label metaclusters from marker medians; embed for display only.

Approach: Median heatmap drives annotation; UMAP colors by cluster but is never used to define or quantify populations.

plotExprHeatmap(sce, features = 'type', by = 'cluster_id', k = 'meta20', scale = 'last')
sce <- runDR(sce, dr = 'UMAP', features = 'type', cells = 2000)
plotDR(sce, dr = 'UMAP', color_by = 'meta20')

7. Differential Abundance and State

Goal: Test which populations change in frequency (DA) or state-marker expression (DS) between conditions.

Approach: The diffcyt() wrapper aggregates to the sample level; results live in res$res. See flow-cytometry/differential-analysis.

design   <- createDesignMatrix(ei(sce), cols_design = 'condition')
contrast <- createContrast(c(0, 1))                        # Treatment vs Control
res_DA <- diffcyt(sce, clustering_to_use = 'meta20', analysis_type = 'DA',
                  method_DA = 'diffcyt-DA-edgeR', design = design, contrast = contrast)
res_DS <- diffcyt(sce, clustering_to_use = 'meta20', analysis_type = 'DS',
                  method_DS = 'diffcyt-DS-limma', design = design, contrast = contrast)
da <- as.data.frame(SummarizedExperiment::rowData(res_DA$res))   # cluster_id, logFC, p_val, p_adj

8. Visualize Results and Export

Goal: Summarize significant populations and persist results.

Approach: Pass the inner result object (res$res) to plotting; export tables and the SCE.

plotDiffHeatmap(sce, res_DA$res, all = TRUE, fdr = 0.05)
plotAbundances(sce, k = 'meta20', by = 'cluster_id', group_by = 'condition')
write.csv(da, 'da_results.csv', row.names = FALSE); saveRDS(sce, 'cytometry_analysis.rds')

Paired / Repeated-Measures Variant

Goal: Account for within-subject correlation (pre/post on the same donor).

Approach: Use a GLMM with a random effect for subject (NOT voom, which is fixed-effects only).

formula <- createFormula(ei(sce), cols_fixed = 'condition', cols_random = 'patient_id')
res_DA <- diffcyt(sce, clustering_to_use = 'meta20', analysis_type = 'DA',
                  method_DA = 'diffcyt-DA-GLMM', formula = formula, contrast = createContrast(c(0, 1)))

Manual Gating Path (alternative to clustering)

Goal: Define populations by a reproducible hierarchy when they are well-defined or rare.

Approach: Build a GatingSet on transformed data; recompute after adding gates. See flow-cytometry/gating-analysis.

library(flowWorkspace)
tl <- estimateLogicle(fs_comp[[1]], colnames(spill))
gs <- GatingSet(transform(fs_comp, tl))
# add openCyto template or manual gates (time -> debris -> singlets -> live -> lineage), then:
recompute(gs); gs_pop_get_stats(gs, type = 'count')

Python Alternative (FlowKit) -- partial

Goal: Read, compensate, and gate in Python where an R pipeline is not an option.

Approach: FlowKit covers IO/compensation/GatingML; there is NO Python equivalent for diffcyt DA/DS, so the differential step stays in R (or bridge via readfcs -> AnnData -> scanpy for clustering only).

import flowkit as fk
sample = fk.Sample('sample.fcs')
sample.apply_compensation(sample.metadata['spillover'])    # FlowKit lowercases + strips $ from keys, so $SPILLOVER -> 'spillover' (not 'spill'); use FlowKit's API, not a hand-rolled inverse
df = sample.as_dataframe(source='comp')

Per-Stage Failure Modes

Per-cell pseudoreplication

Trigger: testing across all cells. Mechanism: cells are not independent replicates. Symptom: p ~ 1e-40 from few subjects. Fix: diffcyt aggregates to sample level; require >= 2-3 replicates/group.

Clustering on state markers

Trigger: activation/phospho markers in features. Mechanism: state contaminates lineage identity. Symptom: activated/resting splits of one type. Fix: cluster on type; test state in DS.

Doublets / wrong cofactor / uncompensated input

Trigger: skipping doublet removal, cofactor 5 on fluorescence, or clustering raw data. Mechanism: phantom double-positives, compressed dim markers, spillover-dominated distances. Symptom: non-reproducible "novel" populations. Fix: remove doublets first; cofactor 5 (CyTOF) / 150 (fluorescence); compensate+transform before clustering.

Batch cleaned instead of modeled

Trigger: CytoNorm-ing then testing naively, or batch confounded with condition. Mechanism: over-correction / non-identifiability. Symptom: attenuated or fabricated effects. Fix: model batch in the design; if batch == condition, no rescue.

Quantitative Thresholds

Common Errors

References

• Weber 2019 Commun Biol 2:183 — diffcyt DA/DS framework.
• Nowicka 2017 F1000Research 6:748 — CATALYST CyTOF workflow; type/state, cofactor 5.
• Weber & Robinson 2016 Cytometry A 89(12):1084-1096 — FlowSOM clustering benchmark.
• Van Gassen 2020 Cytometry A 97(3):268-278 — CytoNorm cross-batch normalization.
• Hurlbert 1984 Ecol Monogr 54(2):187-211 — pseudoreplication (sample is the unit).

Related Skills

• flow-cytometry/fcs-handling - Read FCS and map channels
• flow-cytometry/compensation-transformation - Compensate/unmix and transform
• flow-cytometry/cytometry-qc - Time/margin/dead-cell QC
• flow-cytometry/doublet-detection - Singlet discrimination
• flow-cytometry/bead-normalization - EQ-bead drift and CytoNorm batch correction
• flow-cytometry/gating-analysis - Hierarchical/automated gating path
• flow-cytometry/clustering-phenotyping - FlowSOM clustering and annotation
• flow-cytometry/differential-analysis - diffcyt DA/DS testing
• single-cell/clustering - Related graph-clustering for scRNA-seq