Reactive lymphocytosis can be difficult to differentiate from clonal lymphocytosis, especially when it involves CD8 positive T-lymphocytes. Leukemias and lymphomas cause an increase in this population at the expense of replication of clonal cells with genetic and/or phenotypic aberrations. Infections, mainly of viral etiology, often cause an exacerbated response of this population. The expansion resulting from a viral infection is not selective, so multiple cell clones are involved. Defining the cause of lymphocytosis is essential for determining therapeutic measures.

To define T-cell clonality in patients with peripheral blood lymphocytosis using TCR Vβ2 repertoire by flow cytometry multiparametric analysis (FCM).

We included five patients with suspected T-cell lymphocytosis, one patient with immunodeficiency, and one control. Analysis of TCR Vβ2 rearrangements was performed on whole blood sample using the IOTest Beta Mark TCR Vβ Repertoire Kit (IOTest, Beckman Coulter, Indianapolis, USA) antibody set. BD FACSCantoTM II Cytometer and InfinicytTM 2.0 analysis software were used.

Patient 1 was considered as negative control (lymphocytes 3.41x103/μL) showed expression of various Vβ2 clones, ranging from 0.05 to 1.40%. Patients 2 and 3, both increased CD8 T-lymphocytes associated with a history of infectious disease, had a polyclonal profile of TCR Vβ2 expression, Patient 4 had lymphocytosis (4.25x103/μl) with majority expression of Vβ5.1 clones on CD4 (10.84%) and CD8 (10.56%) lymphocytes and of Vβ 13.2 clone on CD4 lymphocytes (11.53%). This patient was considered with reactive oligoclonal proliferation. Patient 5 had lymphocytosis with an abnormal CD8 T cell phenotype suggestive of Large Granular T-Cell Leukemia. TCR rearrangement analysis confirmed the presence of the Vβ 13.1 clone in CD8 T-cells. Patient 6 had a large lymphocytosis (462.92x103/μL) with a predominance of CD8 positive T-cells and a phenotype suggestive of Prolymphocytic T Cell Leukemia. TCR rearrangement indicated the majority presence of the Vβ8 clone on CD8 T-cell. And finally, patient 7 had mild lymphocytosis (6.87x103/μL) with a predominance of CD8 T-cells with expression of clone Vβ 4 (8.90%) and Vβ17 (2.01%) and of clone Vβ3 on CD4 cells (6.51%) and was considered as oligoclonal expansion.

The confirmation of monoclonal expansion of CD8 T lymphocytes could help in the diagnosis of T-cell neoplasms, confirming the diagnosis of large granular T-cell leukemia and CD8 T-cell prolymphocytic leukemia in two patients.  Among the patients with polyclonal or oligoclonal expression, all patients had a history of viral infectious diseases, such as CMV or EBV infection. Patient 7 had a diagnosis of immunodeficiency with oligoclonal T expansion that can be discussed separately.

Patients presenting with CD8 positive T lymphocytosis and often nonspecific clinical symptoms may benefit from combined evaluation of phenotype and clonal expression by the Vβ2 repertoire.