Detailed analysis of <scp>TRBC1</scp> distribution in T‐cell subsets and its application in T‐cell clonality assessment

Abstract 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.