Semiautomated detection of constant beta chain–restricted CD4⁺ T-cell clusters rapidly identifies Sézary cells, with improved specificity over traditional gating strategies. (A) Blood samples from healthy donors (left), patients with benign inflammatory dermatoses (middle), and patients with CTCL (right) were analyzed by flow cytometry using automated clustering and calculation of percentage TRBC1-positive events per cluster. Individual CD4⁺ T-cell clusters (dots) were classified as clonal (red) or nonclonal (blue) based on pre-established clonality thresholds (dotted lines) or monophasic TRBC1-dim expression on visual analysis. (B) Absolute numbers of Sézary cells (clonal CD4⁺ T cells) per microliter in patients with CTCL. (C) Results of blood ratings based on total clonal CD4⁺ T cells. (D) Number of clonal CD4⁺ T-cell clusters identified per sample on patients with CTLC. (E) Absolute numbers of Sézary cells estimated by clustering and TRBC1 expression compared to the gating strategy recommended by the EORTC. (F) Blood ratings estimated by the EORTC-recommended gating strategy compared to automated clustering and TRBC1 expression. (G) Diagnostic specificity for B1-B2 blood rating (%B0) and analytical specificity (limit of blank) of the EORTC-recommended gating strategy (left) compared to automated clustering and TRBC1 staining (right); based on patients with inflammatory dermatoses.