bio-flow-cytometry-gating-analysis

## Version Compatibility Reference examples tested with: flowCore 2.14+ 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. # Gating Analysis **"Gate my flow cytometry data to identify cell populations"** → Define cell populations through manual or automated gating strategies using rectangular, polygon, or data-driven gates in a hierarchical framework. - R: `flowWorkspace::gs_add_gating_method()`, `openCyto::gating()` for automated gating ## Manual Rectangular Gates ```r library(flowCore) # Create rectangular gate cd4_gate <- rectangleGate(filterId = 'CD4+', 'CD4' = c(500, Inf), 'CD3' = c(200, Inf)) # Apply gate cd4_result <- filter(fcs, cd4_gate) summary(cd4_result) # Get cells in gate cd4_cells <- Subset(fcs, cd4_gate) ``` ## Polygon Gates ```r # Define polygon vertices vertices <- matrix(c(100, 100, # x1, y1 1000, 100, # x2, y2 1000, 1000, # x3, y3 100, 1000), # x4, y4 ncol = 2, byrow = TRUE) colnames(vertices) <- c('FSC-A', 'SSC-A') # Create polygon gate poly_gate <- polygonGate(filterId = 'Lymphocytes', .gate = vertices) # Apply lymph <- Subset(fcs, poly_gate) ``` ## Gating Hierarchy (flowWorkspace) ```r library(flowWorkspace) # Create GatingSet from flowSet gs <- GatingSet(fs) # Add gates to hierarchy gs_pop_add(gs, cd4_gate, parent = 'root') # Add child gate cd4_cd8_gate <- rectangleGate(filterId = 'CD8+', 'CD8' = c(500, Inf)) gs_pop_add(gs, cd4_cd8_gate, parent = 'CD4+') # View hierarchy gs_get_pop_paths(gs) # Recompute statistics recompute(gs) # Get population statistics gs_pop_get_stats(gs) ``` ## Automated Gating: flowDensity ```r library(flowDensity) # Data-driven gate based on density cd4_gate <- deGate(fcs, channel = 'CD4', use.upper = TRUE) # Get threshold cd4_threshold <- cd4_gate@min # Apply cd4_pos <- flowDensity(fcs, channels = 'CD4', position = c(TRUE)) cd4_cells <- getflowFrame(cd4_pos) ``` ## Automated Gating: openCyto **Goal:** Apply a reproducible, template-driven gating strategy that automatically identifies cell populations across all samples. **Approach:** Define a CSV gating template specifying parent-child hierarchy, channel combinations, and gating algorithms (flowClust, singletGate, mindensity, quadrantGate), then apply the template to a GatingSet for batch processing. ```r library(openCyto) # Define gating template gating_template <- fread(' alias,pop,parent,dims,gating_method,gating_args nonDebris,+,root,FSC-A,flowClust,K=2 singlets,+,nonDebris,"FSC-A,FSC-H",singletGate, lymph,+,singlets,"FSC-A,SSC-A",flowClust,K=3 cd3,+,lymph,CD3,mindensity, cd4,+,cd3,"CD4,CD8",quadrantGate, ') # Apply template gt <- gatingTemplate(gating_template) gs <- GatingSet(fs) gating(gt, gs) ``` ## Quadrant Gates ```r # Create quadrant gate quad_gate <- quadGate(filterId = 'CD4_CD8_quad', 'CD4' = 500, 'CD8' = 500) # Results in 4 populations: # CD4+CD8-, CD4-CD8+, CD4+CD8+, CD4-CD8- ``` ## Boolean Gates ```r # Combine gates with logic cd4_not_cd8 <- cd4_gate & !cd8_gate # Alternative using GatingSet gs_pop_add(gs, booleanFilter(CD4+CD8- = CD4+ & !CD8+), parent = 'lymph') ``` ## Extract Gated Populations ```r # Get data for specific population cd4_data <- gh_pop_get_data(gs[[1]], 'CD4+') # Get indices cd4_indices <- gh_pop_get_indices(gs[[1]], 'CD4+') # Counts gs_pop_get_count_fast(gs) ``` ## Visualization ```r library(ggcyto) # Plot with gates autoplot(gs[[1]], 'CD4+') # Multiple populations autoplot(gs[[1]], c('CD4+', 'CD8+')) # Gate overlay autoplot(fcs, 'CD4', 'CD8') + geom_gate(cd4_gate) ``` ## Export Gating Strategy ```r # Save GatingSet save_gs(gs, 'gating_set') # Export to FlowJo workspace library(CytoML) gatingset_to_flowjo(gs, 'analysis.wsp') ``` ## Related Skills - compensation-transformation - Preprocess before gating - clustering-phenotyping - Unsupervised alternative - differential-analysis - Compare gated populations