Flow Cytometric Assessment of the T-Cell Receptor Vβ Repertoire Identifies Clonal T-Cell Populations with High Specificity and Sensitivity.

Multiparameter flow cytometry (MFC) identifies mature T-cell neoplasms based on detection of populations with aberrant expression of T-cell antigens or on the recognition of largely expanded T-cell populations with a regular T-cell immunophenotype. In cases with equivocal findings the differentiation between clonal T-cell populations and reactive conditions is not possible and molecular genetic assessment of T-cell receptor (TCR) rearrangements is needed to prove clonality. The flow cytometric analysis of the TCR Vβ repertoire of T-cell populations has been suggested to improve diagnostics in this regard.

To assess the diagnostic capabilities of the flow cytometric analysis of the TCR Vβ repertoire of T-cell populations in identifying clonal T-cell populations.

We studied 83 patients with a differential diagnosis of mature T-cell neoplasms (29 female, 54 male; median age 65.8 yrs, range 18.4–86.3; peripheral blood n=52, bone marrow n=31). The repertoire of the Vβ chain was analyzed by applying the IOTest® Beta Mark Kit (Beckman Coulter, Miami, FL). This kit contains monoclonal antibodies against 24 different members of variable regions of the TCRβ chain covering about 70% of all members. Vβ analysis was performed 1) for the complete T-cell population of the respective samples identified by CD45-side scatter gating and detection of the expression of pan-T-cell antigens and 2) for T-cell subpopulation identified by an aberrant pattern of T-cell antigen expression. Results were validated using molecular genetic findings on TCR rearrangements as standard: 49 patients had genetically proven TCR rearrangements and 16 patients had not while in 18 patients only a small T-cell clone within a polyclonal background was detected.

The median percentage of expression of the different Vβ members per patient was 1.1% (range, 0% to 99.3%). While the respective median expression values were similar for the different Vβ members ranging from 0.4% to 4.1% the maximum values for the different Vβ members varied considerably between 1.9% and 99.3%. Thus, this data suggests that Vβ members with low expression only, e.g. Vβ11 (range 0% to 1.9%) and Vβ4 (range 0% to 4.8%), were not expressed in any clonal T-cell population in the present cohort while other Vβ members with high expression in some patients clearly were, e.g. Vβ17, Vβ9 and Vβ8 with maximum percentages of 99.3%, 98.3% and 97.8%, respectively. 65 patients with clear-cut genetic results (49 clonal, 16 non-clonal) were subject of validating the use of MFC to identify T-cell clonality. The first step was performed applying a cut-off of 16% for any Vβ member which was found to be the optimum cut-off level. A Vβ member expression >16% was found in 33 cases all of which with were proven clonal by genetics. However, 16 out of the remaining 32 patients with no Vβ member expressed at >16% were also proven clonal by genetics and thus were missed by this first MFC step. Since only about 70% of Vβ members are detected by the applied kit a large clonal T-cell population expressing an undetected Vβ member would result in reduced percentages for the detected Vβ members. Therefore, the second MFC step to identify the presence of clonal T-cell populations was based on the sum of the percentages for all analyzed Vβ members. In 14 of the remaining 32 patients this sum amounted to <58% (suggestive of clonal T-cells expressing a Vβ member not detected by the applied kit) and all 14 were proven clonal by genetics. In the other 18 of the remaining 32 patients this sum was >58%: 16 were proven non-clonal by genetics and 2 were proven clonal by genetics. Thus, overall 47 cases were identified as clonal by MFC with 100% specificity while 2 genetically positive cases were not identified by MFC resulting in a sensitivity of 95.9%. Interestingly, within the 18 cases with only small T-cell clones within a polyclonal background by genetics 6 and 8 were identified positive by MFC using step 1 and 2, respectively, as defined above. The remaining 4 cases revealed subpopulations of T-cells with aberrant immunophenotypes featuring a high expression of single Vβ members (range 26.0 to 88.9%).

The flow cytometric assessment of the T-cell receptor Vβ repertoire identifies clonal T-cell populations with high specificity and sensitivity. It can be performed very easily and quickly and may substitute molecular genetic testing in many cases.

Kern:Beckman Coulter, Krefeld, Germany: Research Funding; MLL Munich Leukemia Laboratory: Equity Ownership. Bellos:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.