End-to-end flow, spectral, and mass cytometry (CyTOF) pipeline from raw FCS files to differentially abundant/expressed cell populations. Orchestrates the read -> compensate/unmix -> transform -> QC -> doublet-removal -> cluster-or-gate -> annotate -> diffcyt DA/DS chain with flowCore/CATALYST/diffcyt, branching on instrument type and on clustering-vs-gating. Use when processing a cytometry experiment end-to-end, deciding the pipeline path for an instrument, or wiring the flow-cytometry component skills into one analysis with valid sample-level statistics.
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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:
packageVersion('<pkg>') then ?function_name to verify parameterspip show <package> then help(module.function) to check signaturesIf code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt rather than retrying. Each stage defers depth to its component skill.
"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.
flowCore + CATALYST::prepData/cluster/runDR + diffcyt::diffcyt()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 load-bearing 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 OPPOSITE ends of the pipeline - EQ-bead drift correction on raw counts at the very front (CyTOF), and CytoNorm cross-batch harmonization after clustering - and conflating them is a classic error.
| Situation | Path | Why |
|---|---|---|
| Conventional fluorescence flow | compensate ($SPILLOVER/flowStats) -> logicle -> ... | optical spillover; logicle handles negatives |
| Spectral cytometer (Aurora/ID7000) | UNMIX (not compensate) -> arcsinh ~150 | overdetermined system; fluorescence-scale |
| Mass cytometry (CyTOF) | EQ-bead normalize (raw) -> arcsinh cofactor 5 -> compCytof if needed | metals barely spill (~1-4%); drift correction first |
| High-dim discovery, no prior gates | cluster (FlowSOM via CATALYST) | scales; finds unexpected populations |
| Well-defined populations / rare events (MRD) | hierarchical gating (openCyto) | interpretable; clustering fails for ultra-rare |
| Multi-batch / multi-day | anchor sample per batch -> CytoNorm (after clustering) | model batch in the design for inference |
FCS -> compensate/unmix -> transform -> QC (margins, time, dead) -> doublets
-> [ cluster (FlowSOM) | gate (openCyto) ] -> annotate -> diffcyt DA/DS -> report
EQ-bead drift normalization (CyTOF) runs on raw counts BEFORE everything; CytoNorm runs LAST.
Goal: Define the type/state panel and sample metadata, then load FCS.
Approach: Panel marker_class drives everything downstream (type clusters, state is tested); metadata keys samples to condition/subject. See flow-cytometry/fcs-handling.
library(CATALYST); library(diffcyt); library(flowCore); library(ggplot2)
panel <- data.frame(
fcs_colname = c('FSC-A','SSC-A','CD45','CD3','CD4','CD8','CD19','CD14','Ki67','IFNg'),
antigen = c('FSC','SSC','CD45','CD3','CD4','CD8','CD19','CD14','Ki67','IFNg'),
marker_class = c('none','none','type','type','type','type','type','type','state','state'))
md <- data.frame(file_name = list.files('data', pattern = '\\.fcs$'),
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)
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.
fs_comp <- compensate(fs, spillover(fs[[1]])[[1]]) # 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)
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'); plotMDS(sce, color_by = 'condition')
# event-level cleaning runs per-FCS upstream: PeacoQC::RemoveMargins() -> PeacoQC()/flowAI on transformed data
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]
}
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)
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')
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$res)) # cluster_id, logFC, p_val, p_adj
Goal: Summarize significant populations and persist results.
Approach: Pass the inner result object (res$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')
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)))
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(spillover(fs[[1]])[[1]]))
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')
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['spill']) # use FlowKit's API, not a hand-rolled matrix inverse
df = sample.as_dataframe(source='comp')
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.
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.
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.
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.
| Threshold | Source | Rationale |
|---|---|---|
| arcsinh cofactor 5 (CyTOF) / ~150 (fluorescence) | Nowicka 2017 F1000Res 6:748 | matches platform noise scale |
| >= 2-3 biological replicates per group | Weber 2019 Commun Biol 2:183 | minimum for a valid DA/DS error term |
| > ~10K cells per sample | community | stable per-sample cluster frequencies |
| 10-30 metaclusters typical (maxK=20 default) | Weber & Robinson 2016 Cytometry A 89:1084 | over-provision then merge |
| BH FDR across clusters (and clusters x markers for DS) | diffcyt | many simultaneous tests |
| Error / symptom | Cause | Solution |
|---|---|---|
testDA_edgeR(sce, ...) not found / wrong | fabricated signature | use the diffcyt() wrapper; results in res$res |
compensate() errors on a list | spillover(ff) returns a list | index [[1]] |
| empty DS results | state markers not flagged | set marker_class='state' in the panel |
| paired design ignored | used fixed-effect method | diffcyt-DA-GLMM with a random effect |
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