Combined Flow Cytometric Approach for the Characterization of Canine Small Cell Lymphomas

Flow cytometry has emerged as a reliable tool for diagnosis of canine lymphoma, contributing to the evaluation of cell size, lineage, clonality, and staging. In clinical practice lymphomas are classified mainly by the use of cytology. However, lymph node aspirates without corresponding smears are often sent to laboratories and the final diagnosis is reached only by flow cytometric data. In these cases, flow immunophenotyping is sufficient to identify B- or T-high grade lymphomas associated with large sized cells, while flow cytometry can only provide cell lineage when tumors are associated with small or medium sized cells. The aim of this study was to characterize canine small/medium sized cell lymphomas (SML) with diagnostic purposes, through the flow cytometric determination of proliferative and apoptotic activity. Fifty-eight SML were diagnosed based on flow cytometric immunophenotyping and scatter properties (FSC) were included. According to the updated Kiel classification these were 27 high grade (HG) forms (9 B-cell, 18 T-cell) and 31 low grade (LG) forms (11 B-cell, 20 T-cell). Proliferation and apoptosis were determined on lymph node aspirates using, respectively, an anti-Ki67-FITC monoclonal antibody (clone MIB-1) and an AnnexinV-FITC/Propidium Iodide Kit. Proliferative activity was expressed as percentage of Ki67-positive cells (Ki67%), while apoptotic activity was expressed as percentage of AnnexinVpositive/ PI-negative cells (AnnV+%). Moreover, a proliferation/apoptosis ratio (PAR) was calculated. Differences in Ki67%, AnnV+% and PAR between and within groups were assessed by t-test or Mann-Whitney test. Ki67% was significantly higher (p < 0.001) in HG (37.6% ± 14.4) with respect to LG lymphomas (5.6% ± 3.2), while AnnV+% was significantly higher (p < 0.001) in B-cell (56.18% ± 18.61) with respect to T-cell lymphomas (13.02% ± 9.39). Therefore, PAR was significantly higher (p < 0.001) in HG (3.19% ± 3.45) with respect to LG lymphomas (0.45% ± 0.53) and, within each malignancy grade, it was significantly higher (p < 0.001) in T-cell respect to B-cell forms. Decreasing values were recorded in HG T-cell (4.13% ± 3.66), HG B-cell (HG = 1.32% ± 2.07), LG T-cell (0.62% ± 0.6), and LG B-cell (0.14% ± 0.06) lymphomas. In particular, PAR clearly differentiated macronucleolated (0.13% ± 0.06) from lymphoblastic (1.61% ± 2.29) lymphomas, among B-cell tumors, and pleomorphic mixed cell (2.87% ± 1.97) from small clear cell (0.34% ± 0.16) lymphomas, among T-cell tumors. Ki67% reflects malignancy grade, AnnV+% is associated with cell lineage and PAR is able to characterize different types of lymphomas. Thus, a combined flow cytometric evaluation of FSC, immunophenotype, proliferative, and apoptotic activity may represent a useful tool to classify canine SML, regardless of cytology. However, cytologic examination remains mandatory for the correct definition of specific Kiel subtypes.