Exclusion of cytoplasmic fragments in flow cytometric analysis of lymph node samples from dogs with lymphoma using membrane-permeable violet laser-excitable DNA-binding fluorescent dye (DyeCycle Violet)

Background: Cytoplasmic fragments derived from fragile neoplastic lymphocytes are common in samples of lymph nodes collected from dogs with lymphoma. These cytoplasmic fragments interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membranes. Objective: The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments from flow cytometric analysis of canine lymph nodes in which lymphoma was present. Methods: Single‐cell suspensions of neoplastic cells were prepared from biopsy samples and fine‐needle aspirates of lymph nodes from 23 dogs with lymphoma. Suspensions were stained using a violet laser‐excitable (405 nm) membrane‐permeable DNA‐binding fluorescent dye (DyeCycle Violet DCV]), incubated with antibodies against CD3, CD5, CD21, CD22, and CD45, and then stained with 7‐amino‐actinomycin D (7‐AAD), an argon‐excitable (488 nm) membrane‐impermeable DNA‐binding fluorescent dye. Multiparameter flow cytometry was used for analysis based on selective uptake and laser‐activated fluorescence of these dyes. Results: Cytoplasmic fragments, which were DCV‐negative and CD45‐positive, and dead cells, which were positive for 7‐AAD, were efficiently separated from neoplastic cells. Conclusion: Staining with DCV is a useful method to improve flow cytometric gating methods and quantitative analyses of lymph node samples from dogs with lymphoma.