Exclusion of cytoplasmic fragments in flow cytometric analysis of lymphoma samples using DyeCycle Violet (144.1)

Abstract

Abstract Cytoplasmic fragments that originate from fragile lymphoid cells and are commonly seen in lymphoma fine needle aspirate samples. These cytoplasmic fragments can 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 membrane. The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments in lymphoma samples analyzed by flow cytometry. Tumor cells prepared from lymph node biopsy and fine needle aspirate samples of dogs with lymphoma were stained using DyeCycle Violet (DCV, a membrane-permeable DNA-binding fluorescent dye), antibodies against cell surface antigens, and 7-amino-actinomycin D (7-AAD, a membrane impermeable, DNA-binding fluorescent dye). Multi-parameter flow cytometry was used to exclude antigen-positive cytoplasmic fragments for analysis based on the selective uptake and fluorescence of these dyes. Both cytoplasmic fragments, which are DCV-negative and CD45-positive, and dead cells, which are 7-AAD-positive, could be efficiently separated from tumor cells in lymphoma samples. DCV staining is a useful method to improve the gate setting and quantitative analyses of lymphoma samples by flow cytometry. DI and JFM contributed equally to this work