Yang M H, Lin S J
Department of Pediatrics, Chang Gung Children's Hospital, Taoyuan, Taiwan.
Pediatr Hematol Oncol. 2001 Jan-Feb;18(1):57-63. doi: 10.1080/088800101750059864.
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.
通过Ficoll-Hypaque密度梯度离心法（FDGC）获得的脐带血（CB）单个核细胞（MNC）常被红细胞和有核红细胞前体污染。作者研究了两轮（2-r）FDGC对CB和成人外周血（APB）MNC淋巴细胞亚群和自然杀伤活性的影响，并与传统的一轮（1-r）分离法获得的结果进行比较。在第二次密度梯度离心步骤后，表达CD45的CB MNC百分比大幅增加（p <.01），表明纯化效果良好。与一轮分离后相比，两轮FDGC后CD3/CD4、CD3/CD8和CD16/56双阳性染色的CB MNC百分比显著增加。然而，两轮FDGC对CD34 +干细胞和CD19 + B细胞的百分比没有影响。两轮FDGC获得的MNC比一轮分离获得的MNC具有更高的自然杀伤（NK）活性（p <.01）。相比之下，与一轮FDGC相比，两轮FDGC对APB MNC的淋巴细胞亚群和NK活性没有影响。因此，推荐使用两轮FDGC来获取用于流式细胞术分析和NK细胞毒性测定的CB MNC。
