Dual T-cell constant β chain (TRBC)1 and TRBC2 staining for the identification of T-cell neoplasms by flow cytometry

The diagnosis of leukemic T-cell malignancies is often challenging, due to overlapping features with reactive T-cells and limitations of currently available T-cell clonality assays. Recently developed therapeutic antibodies specific for the mutually exclusive T-cell receptor constant β chain (TRBC)1 and TRBC2 isoforms provide a unique opportunity to assess for TRBC-restriction as a surrogate of clonality in the flow cytometric analysis of T-cell neoplasms. To demonstrate the diagnostic utility of this approach, we studied 135 clinical specimens with (50) or without (85) T-cell neoplasia, in addition to 29 blood samples from healthy donors. Dual TRBC1 and TRBC2 expression was studied within a comprehensive T-cell panel, in a fashion similar to the routine evaluation of kappa and lambda immunoglobulin light chains for the detection of clonal B-cells. Polytypic TRBC expression was demonstrated on total, CD4⁺ and CD8⁺ T-cells from all healthy donors; and by intracellular staining on benign T-cell precursors. All neoplastic T-cells were TRBC-restricted, except for 5 cases (10%) lacking TRBC expression. T-cell clones of uncertain significance were identified in 15 samples without T-cell malignancy (13%), and accounted for smaller subsets than neoplastic clones (median: 4.7% vs. 73% of lymphocytes, p<0.0001). Single staining for TRBC1 produced spurious TRBC1-dim subsets in 21 clinical specimens (16%), all of which resolved with dual TRBC1/2 staining. Assessment of TRBC restriction by flow cytometry provides a rapid diagnostic method to detect clonal T-cells, and to accurately determine the targetable TRBC isoform expressed by T-cell malignancies.