Detailed analysis of TRBC1 distribution in T-cell subsets and its application in T-cell clonality assessment

Assessment of T-cell clonality is critical for diagnosing T-cell non-Hodgkin lymphomas (T-NHL). Flow cytometric T-cell receptor (TCR)-Vβ-repertoire analysis is specific but technically complex, multi-tube, and often limited in paucicellular specimens. T-cell receptor-beta-constant-region-1 (TRBC1) has emerged as a simpler alternative, but normal distribution ranges in clinically relevant T-cell subsets remain insufficiently characterized and data on direct comparison with TCR-Vβ restriction is limited. We prospectively analyzed 70 blood samples from healthy donors and 58 samples (bone marrow, peripheral blood, fine-needle aspirates, body fluids) from T-NHL patients. Multiparameter flow cytometry (MFC) was used for diagnostic immunophenotyping in T-NHL cases and for TRBC1 assessment in T-NHL cases and 28 immunophenotypic T-cell subsets in healthy individuals. In T-NHL, clonality was determined by TRBC1 restriction in aberrant T-cell populations and, where sufficient material was available, corroborated by TCR-Vβ repertoire analysis (IOTest Beta Mark).In healthy donors, median TRBC1-positive fractions across T-cell subsets ranged from 21.39% to 78.61%, with narrow 99% confidence intervals in major CD4⁺ and CD8⁺ compartments. Minor subsets occasionally showed extreme values (7%-93%) but remained polyclonal by TCR-Vβ analysis, leading to thresholds of ≤6% or ≥94% TRBC1 positivity as indicative of clonality in our cohort. Among 57 evaluable T-NHL cases, all CD3-positive neoplastic populations showed monotypic TRBC1 expression, with 100% concordance with TCR-Vβ results in 18 tested cases. TRBC1 was non-informative on surface-CD3-negative T-NHL, although cytoplasmic staining was sometimes helpful. Flow cytometric TRBC1 analysis is a rapid, cost-effective, single-tube method for T-cell clonality assessment, with applicability to small T-cell subsets such as paucicellular and measurable residual disease samples.