Anti-TRBC1 Antibody-Based Flow Cytometric Detection of T-Cell Clonality: Standardization of Sample Preparation and Diagnostic Implementation

SIMPLE SUMMARY: The anti-TRBC1 antibody JOVI-1 has recently been identified as a flow cytometry marker potentially useful for assessment of T-cell clonality. The aim of this study was to optimize a flow cytometric method for routine use of anti-TRBC1 to assess T-cell clonality and validate it in a large series of normal and pathological samples. Our results showed that the best resolution to accurately identify TRBC1(+) cells was achieved by adding the CD3 antibody either simultaneously or after TRBC1. In addition, TRBC1(+)/TRBC1(−) ratios within different Tαβ-cell subsets are provided as expected reference ranges for polyclonal T-cells. Based on the optimized approach here proposed, we detected monoclonal Tαβ-cell populations with high specificity (96%) and a high analytical sensitivity/level of detection (≤10(−4)), when clonal T-cells exhibited immunophenotypic aberrancies. These findings further support and extend previous observations about the utility of TRBC1 for the diagnostic screening and monitoring of clonal Tαβ-cell populations. ABSTRACT: A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1(+) and TRBC1(−) monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1(+)/TRBC1(−) ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ families, whereas highly-variable TRBC1(+)/TRBC1(−) ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10(−4) in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ(+) leukemia/lymphoma patients.