Flow cytometric immunophenotypic assessment of T-cell clonality by vβ repertoire analysis in fine-needle aspirates and cerebrospinal fluid

Abstract

Flow cytometric T-cell receptor V(β) repertoire analysis (TCR-V(β)-R) is a sensitive method to detect T-cell clonality; however, its implementation in low-cellularity specimens has not been established. We developed a strategy to use TCR-V(β)-R in cerebrospinal fluid (CSF) and fine-needle aspirate (FNA) specimens. Initially, full TCR-V(β)-R was evaluated in diagnostic/screening specimens from 8 patients with T-cell neoplasia to determine tumor-specific TCR-V(β) protein expression. Subsequently, an abbreviated, patient-specific TCR-V(β)-R evaluation was performed in 17 paucicellular specimens from the patients (8 CSF, 9 FNA) for staging and monitoring of minimal residual disease (MRD). A single cocktail containing 3 anti-V(β) antibodies (1 tumor-specific and 2 negative controls) in combination with other antibodies chosen to help gate on atypical T cells is highly sensitive and specific for detecting low-level neoplastic T-cell involvement in paucicellular specimens. This TCR-V(β)-R strategy is valuable in staging and evaluating MRD in patients with T-cell non-Hodgkin lymphoma.

Image 1

A, B, and C (Case 7), Abnormal T cells in are CD3+ and dim CD8+. An analysis (A, polygon) is drawn on the abnormal T cells for V_β repertoire analysis (B and C) with anti-V_β 7.2 fluorescein isothiocyanate (FITC), anti-V_β 13.2 phycoerythrin (PE), and anti-V_β 4 FITC plus PE (double-labeled). Abnormal T cells are restricted to V_β 4 in blood (B) and cerebrospinal fluid (C). D, E, and F (Case 3), Abnormal T cells are dim CD3+ and CD4+. An analysis (D, polygon) is drawn on the abnormal T cells for V_β repertoire analysis (E and F) with anti-V_β 8 FITC, anti-V_β 13.1 PE, and anti-V_β 13.6 FITC plus PE (double-labeled). Abnormal T cells are restricted to V_β 13.1 in blood (E) and fine-needle aspirate specimen (F). APC, allophycocyanin; PerCP, peridinin-chlorophyll-a protein.