Effect of two-round Ficoll-Hypaque density gradient centrifugation on lymphocyte subsets and natural killer activity of umbilical cord blood mononuclear cells

Umbilical cord blood (CB) mononuclear cells (MNCs) obtained from Ficoll-Hypaque density gradient centrifugation (FDGC) are frequently contaminated with erythrocytes and nucleated erythroid precursors. The authors investigated the effect of two-round (2-r) FDGC on lymphocyte subsets and natural killer activity of CB and adult peripheral blood (APB) MNCs, in comparison with those obtained from conventional one-round (1-r) separation. The percentage of CD45-expressing CB MNCs was greatly increased after the second density step (p < .01), indicating the efficacy in purification. The percentages of CD3/CD4, CD3/CD8, and CD16/56 double-positive staining CB MNCs were significantly increased after 2-r FDGC, as compared to those after 1-r separation. However, the percentages of CD34⁺ stem cells and CD19⁺ B cells were not affected by 2-r FDGC. MNCs obtained from 2-r FDGC had higher natural killer (NK) activity than did MNCs obtained from 1-r separation (p < .01). In contrast, 2-r FDGC did not affect lymphocyte subsets and NK activity of APB MNCs as compared to 1-r FDGC. Thus, 2-r FDGC are recommended to obtain CB MNCs for flow cytometric analysis and NK cytotoxicity assays.