flow-cytometry-clustering-phenotyping

name	flow-cytometry-clustering-phenotyping
description	Unsupervised clustering and cell-type identification for high-dimensional flow, spectral, and mass cytometry - FlowSOM, PhenoGraph, FlowSOM-via-CATALYST, with UMAP/tSNE for visualization. Covers the type-vs-state marker distinction (cluster on lineage, test state within clusters), over-provision-then-metacluster, the Weber-Robinson benchmark, seed dependence and metacluster stability, why embeddings are for looking not measuring, and median-heatmap annotation/merging. Use when discovering populations without predefined gates, choosing a clustering algorithm, selecting the number of metaclusters, or annotating clusters into cell types.
tool_type	r
primary_tool	CATALYST

Version Compatibility

Reference examples tested with: CATALYST 1.26+, FlowSOM 2.10+, flowCore 2.14+; Rphenograph (GitHub: JinmiaoChenLab/Rphenograph).

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

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

Rphenograph is GitHub-only (remotes::install_github('JinmiaoChenLab/Rphenograph')) and returns a list - membership is igraph::membership(out[[2]]), not a vector. Adapt rather than retrying.

Clustering and Phenotyping

"Cluster my cytometry data to find cell types" -> Discover populations in high-dimensional data without gates, then annotate them by marker expression.

• R: CATALYST::cluster() (wraps FlowSOM + ConsensusClusterPlus) - the field default
• R: FlowSOM::FlowSOM() directly, or Rphenograph() for graph-based clustering

The Single Most Important Modern Insight -- Cluster on Type Markers; the Embedding Is for Looking, Not Measuring

Two rules carry most of the correctness here. First, the type-vs-state distinction: LINEAGE/type markers (CD3, CD4, CD8, CD19) DEFINE clusters; functional/STATE markers (phospho-epitopes, cytokines, Ki-67, activation markers) must be WITHHELD from clustering and tested within clusters instead (the DA/DS framework, Nowicka 2017 F1000Res 6:748). Clustering on state markers splits "activated CD4" from "resting CD4" and confounds abundance with activation - a classic, silent design error. Second, t-SNE/UMAP embeddings do NOT preserve inter-cluster distances, cluster sizes, or densities (the apparent "UMAP preserves global structure" edge over tSNE is largely an initialization artifact - Kobak & Linderman 2021 Nat Biotechnol 39:156). Define populations by clustering in the HIGH-DIMENSIONAL space and COLOR the embedding by cluster; never gate on the embedding or read biology off blob distances.

Algorithm Taxonomy

Algorithm	Citation	Mechanism	Speed	Rare-pop	Determinism
FlowSOM	Van Gassen 2015 Cytometry A 87:636	SOM grid -> MST (viz) -> consensus metaclustering	fastest	good if grid over-provisioned	stochastic; seed-controllable
PhenoGraph	Levine 2015 Cell 162:184	kNN graph (Jaccard) + Louvain	moderate	strong (no preset k)	seed-fragile (>40% reassignment reported)
X-shift	Samusik 2016 Nat Methods 13:493	weighted kNN density + auto cluster #	slow	excellent	more deterministic
flowMeans	Aghaeepour 2011 Cytometry A 79:6	k-means multi-cluster + change-point k	fast	moderate	stochastic

Benchmark: Weber & Robinson 2016 Cytometry A 89:1084 tested 18 methods - FlowSOM (with metaclustering) was a top performer AND by far fastest, hence the field default; but its accuracy depends on supplying the right number of metaclusters.

Why Over-Provision the Grid, Then Metacluster

Set the SOM grid (e.g. 10x10 = 100 nodes) MUCH larger than the number of populations expected, then metacluster down. The asymmetry: metaclustering can MERGE over-fine nodes into a real population, but can NEVER SPLIT a node that erroneously fused two cell types. Too coarse commits the unrecoverable error; too fine commits only the recoverable one. So over-cluster, then merge by hand off the median heatmap.

CATALYST Clustering Pipeline

Goal: Cluster on type markers and prepare for annotation.

Approach: prepData builds the SCE (panel marker_class flags type vs state); cluster() wraps FlowSOM+ConsensusClusterPlus. Defaults xdim=ydim=10, maxK=20 (the metacluster cap people forget); set seed on the function.

library(CATALYST)

sce <- prepData(fs, panel, md, transform = TRUE, cofactor = 5)   # cofactor 5 = CyTOF; ~150 for fluorescence
sce <- cluster(sce, features = 'type',                            # type markers only
               xdim = 10, ydim = 10, maxK = 20, seed = 42)        # maxK caps metaclusters at 20 by default
plotExprHeatmap(sce, features = 'type', by = 'cluster_id', k = 'meta20', scale = 'last')

PhenoGraph (graph-based alternative)

Goal: Cluster with a kNN graph when a data-driven cluster count is wanted.

Approach: Rphenograph on the type-marker matrix (cells x markers); extract membership from the list.

library(Rphenograph)
type_expr <- t(assay(sce, 'exprs')[rowData(sce)$marker_class == 'type', ])
out <- Rphenograph(type_expr, k = 30)                  # only knob: k (neighbors)
sce$phenograph <- factor(igraph::membership(out[[2]]))  # list -> membership, not a vector

Dimensionality Reduction (visualization only) and Annotation

Goal: Visualize structure and assign cell-type labels.

Approach: runDR subsamples per sample (cells=); color by cluster, never gate on it. Annotate from the median heatmap, then mergeClusters with a curated table.

sce <- runDR(sce, dr = 'UMAP', features = 'type', cells = 2000)   # subsampled embedding
plotDR(sce, 'UMAP', color_by = 'meta20')

merging <- data.frame(old_cluster = 1:20,
                      new_cluster = c('CD4 T','CD4 T','CD8 T', '...'))   # curated from the heatmap
sce <- mergeClusters(sce, k = 'meta20', table = merging, id = 'annotated')

Per-Method Failure Modes

Clustering on state markers

Trigger: activation/phospho markers in the clustering feature set. Mechanism: state contaminates lineage identity. Symptom: "activated" and "resting" versions of a type split as separate clusters. Fix: cluster on type only; test state markers within clusters (differential-analysis).

Seed-dependent "novel populations"

Trigger: a population that appears at one seed and vanishes at another. Mechanism: FlowSOM init / Louvain are stochastic. Symptom: non-reproducible clusters. Fix: set + report the seed; check multi-seed stability; treat unstable clusters as hypotheses.

Reading biology off the embedding

Trigger: "cluster A is closer to B than C." Mechanism: UMAP/tSNE distances are non-metric. Symptom: false developmental/relatedness claims. Fix: quantify in marker space; embedding for display only.

Clustering uncompensated/untransformed data

Trigger: raw linear input to FlowSOM. Mechanism: spillover + scale dominate Euclidean distance. Symptom: clusters track intensity, not biology. Fix: compensate + transform first.

Quantitative Thresholds

Threshold	Source	Rationale
over-provision grid (10x10) >> expected pops	Van Gassen 2015	metacluster can merge, never split
maxK = 20 default	CATALYST	metacluster cap; raise if expecting more
FlowSOM needs correct K	Weber & Robinson 2016	accuracy depends on metacluster number
use median (not mean) per cluster	Bendall 2011 Science 332:687	robust to doublet/spillover contamination

Common Errors

Error / symptom	Cause	Solution
clustering uses scatter/Time/state	features not restricted	features='type' / colsToUse= lineage markers
Rphenograph result unusable	it returns a list	igraph::membership(out[[2]])
set.seed doesn't make FlowSOM reproducible	internal reseeding	pass seed= to cluster()
only 20 clusters no matter what	maxK default	raise maxK

References

• Van Gassen 2015 Cytometry A 87(7):636-645 — FlowSOM.
• Levine 2015 Cell 162(1):184-197 — PhenoGraph.
• Samusik 2016 Nat Methods 13(6):493-496 — X-shift.
• Weber & Robinson 2016 Cytometry A 89(12):1084-1096 — clustering benchmark (FlowSOM top + fastest).
• Nowicka 2017 F1000Research 6:748 — CyTOF workflow; type-vs-state markers.
• Kobak & Linderman 2021 Nat Biotechnol 39:156-157 — embedding initialization artifact.
• Bendall 2011 Science 332(6030):687-696 — arcsinh-median analysis of CyTOF data.

Related Skills

• compensation-transformation - Compensate/transform before clustering
• gating-analysis - Supervised alternative; needed for rare populations
• differential-analysis - Test abundance/state of clusters between conditions
• cytometry-qc - Cluster only QC-passed events
• single-cell/clustering - Leiden/Louvain on scRNA-seq (shared graph-clustering ideas)
• imaging-mass-cytometry/phenotyping - Same CATALYST/FlowSOM conventions for imaging