Abstract
Simple SummaryThe 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.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.
Highlights
T-cell chronic lymphoproliferative disorders (T-CLPD) are uncommon lymphoid malignancies derived from post-thymic T-cells [1,2], which comprise a heterogeneous group of entities with variable clinical behavior [1,3] and biologic features [4,5,6,7,8]
When we compared different incubation conditions for the CD3 and TRBC1 double-staining on the TRBC1 expression profile in terms of both stain index (SI) and median fluorescence intensity (MFI) of the TRBC1+ and TRBC1− Tαβ-cell populations (Figure 2D,E), we found that addition of TRBC1 either prior or simultaneously to CD3 was associated with the highest TRBC1 SI on Tαβ-cells and, thereby, a more clear discrimination between TRBC1+ and TRBC1− Tαβ-cells (Figure 2D) was observed, with progressively decreased MFI values of TRBC1+ at the expense of greater (p < 0.05) CD3 MFI values in Tαβ-cells (Figure 2E)
Despite the low number of T-CLPD cases analyzed within each WHO2017 diagnostic category, our results showed a potentially skewed usage of TRBC2 vs. TRBC1 in T-cell prolymphocytic leukemia (T-PLL), that contrast with primary cutaneous T-cell lymphoma (PCTCL)-Sézary syndrome (SS) and HD with a Tαβ-cell clone (HDc), which more frequently involved the TRBC1 region
Summary
T-cell chronic lymphoproliferative disorders (T-CLPD) are uncommon lymphoid malignancies (approximately 10–15% of all peripheral/mature lymphoid neoplasms worldwide) derived from post-thymic T-cells [1,2], which comprise a heterogeneous group of entities with variable clinical behavior [1,3] and biologic features [4,5,6,7,8]. TR gene rearrangement analysis by PCR is relatively complex and time-consuming (requires experienced personnel and results are generally not available on the same day), does not provide accurate quantitation of the size of the T-cell clone, and/or lacks simultaneous information about the phenotypic characteristics of the expanded clone, which needs to be discriminated from the background of polyclonal T-cells [14,15]; sometimes it might even require prior enrichment/isolation of the suspicious clonal T-cell population to reach enough sensitivity [14,16,17] Both FCM and PCR assays are not routinely available in many diagnostic laboratories due to the low prevalence of T-CLPD
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