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Background: Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T-cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells., Methods: Here, we used a single TRBC1 antibody, in conjunction with other T-cell associated markers, to detect T-cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T-cell neoplasm and 97 patients with no T-cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1., Results: We examined TRBC1 expression on neoplastic T-cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T-cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T-cell neoplasms, and arbitrarily dissected T-cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets., Conclusions: Single TRBC1 antibody detection of T-cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories., (© 2020 International Clinical Cytometry Society.)

Introducing a sensitive and specific peripheral blood flow cytometric assay for Sézary syndrome and mycosis fungoides (SS/MF) requires careful selection of assay design characteristics, and translation into a laboratory developed assay through development/optimization, validation, and continual quality monitoring. As outlined in a previous article in this series, the recommended design characteristics of this assay include at a minimum, evaluation of CD7, CD3, CD4, CD8, CD26, and CD45, analyzed simultaneously, requiring at least a 6 color flow cytometry system, with both quantitative and qualitative components. This article provides guidance from an international group of cytometry specialists in implementing an assay to those design specifications, outlining specific considerations, and best practices. Key points presented in detail are: (a) Pre-analytic components (reagents, specimen processing, and acquisition) must be optimized to: (i) identify and characterize an abnormal population of T-cells (qualitative component) and (ii) quantitate the abnormal population (semi/quasi-quantitative component). (b)Analytic components (instrument set-up/acquisition/analysis strategy and interpretation) must be optimized for the identification of SS/MF populations, which can vary widely in phenotype. Comparison with expert laboratories is strongly encouraged in order to establish competency. (c) Assay performance must be validated and documented through a validation plan and report, which covers both qualitative and semi/quasi-quantitative assay components (example template provided). (d) Ongoing assay-specific quality monitoring should be performed to ensure consistency., (© 2020 International Clinical Cytometry Society.)

This review discusses the definition and major categories of cutaneous T-cell lymphoma, Sézary syndrome and mycosis fungoides, and the role of immunophenotyping in their diagnosis. The following key points are raised: (a) Sézary syndrome and mycosis fungoides cells most often have a characteristic CD3+ CD4+ CD7- and/or CD26- immunophenotype. (b) This immunophenotype is not specific, but can assist in the distinction from non-neoplastic T cells and other subtypes of mature T-cell neoplasm. (c) However, small subsets of normal and reactive T-cells can have an overlapping immunophenotype, and can be distinguished by evaluating for additional changes in antigen expression., (© 2020 International Clinical Cytometry Society.)

A peripheral blood flow cytometric assay for Sézary syndrome (SS) or circulating mycosis fungoides (MF) cells must be able to reliably identify, characterize, and enumerate T-cells with an immunophenotype that differs from non-neoplastic T-cells. Although it is also important to distinguish SS and MF from other subtypes of T-cell neoplasm, this usually requires information in addition to the immunophenotype, such as clinical and morphologic features. This article outlines the approach recommended by an international group with experience and expertise in this area. The following key points are discussed: (a) At a minimum, a flow cytometric assay for SS and MF should include the following six antibodies: CD3, CD4, CD7, CD8, CD26, and CD45. (b) An analysis template must reliably detect abnormal T-cells, even when they lack staining for CD3 or CD45, or demonstrate a phenotype that is not characteristic of normal T-cells. (c) Gating strategies to identify abnormal T-cells should be based on the identification of subsets with distinctly homogenous immunophenotypic properties that are different from those expected for normal T-cells. (d) The blood concentration of abnormal cells, based on any immunophenotypic abnormalities indicative of MF or SS, should be calculated by either direct enumeration or a dual-platform method, and reported., (© 2020 International Clinical Cytometry Society.)

Background: The diagnosis of T-cell neoplasms is often challenging, due to overlapping features with reactive T-cells and limitations of currently available T-cell clonality assays. The description of an antibody specific for one of two mutually exclusive T-cell receptor (TCR) β-chain constant regions (TRBC1) provide an opportunity to facilitate the detection of clonal TCRαβ T-cells based on TRBC-restriction., Methods: Twenty patients with mature T-cell neoplasms and 44 patients without evidence of T-cell neoplasia were studied. Peripheral blood (51), bone marrow (10), and lymph node (3) specimens were evaluated by 9-color flow cytometry including TRBC1 (CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1 and/or CD2/CD3/CD4/CD5/CD7/CD8/CD26/CD45/TRBC1). Monophasic TRBC1 expression on any immunophenotypically distinct CD4-positive or CD8-positive/TCRγδ-negative T-cell subset was considered indicative of clonality., Results: Monophasic (clonal) TRBC1 expression was identified on immunophenotypically abnormal T-cells from all 20 patients with T-cell malignancies (100% sensitivity), including 17 cases with either >97% or <3% TRBC1-positive events, and three cases with monophasic homogenous TRBC1-dim expression. All immunophenotypically distinct CD4-positive and CD8-positive/TCRγδ-negative T-cell subsets from 44 patients without T-cell malignancies showed the expected mixture of TRBC1-positive and TRBC-1-negative subpopulations (non-clonal), except for seven patients (16%) with very small CD8-positive T-cell subsets exhibiting a monophasic (clonal) pattern., Conclusion: Inclusion of a single anti-TRBC1 antibody into a diagnostic T-cell flow cytometry panel facilitates the rapid identification of T-cell neoplasms, in addition to small monotypic CD8-positive subsets of uncertain significance. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background: Mycosis fungoides (MF) and Sézary Syndrome (SS) are clinically distinct cutaneous T-cell lymphomas with strikingly similar morphologic and phenotypic features. Prior studies have suggested phenotypic differences based on markers of antigen experience, suggesting a different cell of origin., Methods: Seventy-nine involved peripheral blood or bone marrow samples from 33 patients with SS and 19 patients with MF were studied by 10-color flow cytometry, including CD62L, CD45RA, CCR4, and PD-1. Gated tumor events were classified as naïve (T N ), central memory (T CM ), effector memory (T EM ), or effector memory with reacquired CD45RA (T EMRA ); based on CD62L + /CD45RA + , CD62L + /CD45RA - , CD62L - /CD45RA - , or CD62L - /CD45RA + phenotype, respectively. Sequential specimens were compared to assess for phenotypic stability., Results: The naïve/memory phenotype of the neoplastic T-cells was markedly heterogeneous, with a dominant T N , T CM , T EM , or T EMRA subset on 11 (14%), 32 (41%), 30 (38%), and 6 (8%) cases, respectively. There was no correlation between the diagnosis of MF or SS and putative cell of origin (P = 0.4). Overexpression of CCR4 and PD1 was observed in most cases, with higher intensity in SS compared to MF. The naïve/memory phenotype remained the same for 10 patients up to 273 days after the initial analysis; while on six patients, the naïve/memory phenotype was different from the original phenotype., Conclusions: Both SS and MF can have phenotypic features of any of the major naïve/memory T-cell subsets, which questions the current principle of "cell-of-origin" distinction between SS and MF. Phenotypic shifts within these subsets are common, suggesting a functional state rather than a cell-of-origin surrogate. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)