Irsch J, Nitsch S, Hansmann M L, Rajewsky K, Tesch H, Diehl V, Jox A, Küppers R, Radbruch A
Institute for Genetics, University of Cologne, 50931 Cologne, Germany.
Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):10117-22. doi: 10.1073/pnas.95.17.10117.
Hodgkin disease (HD) is characterized by a small number of malignant Hodgkin and Reed-Sternberg (H/RS) cells among a major population of nonmalignant cells. The analysis of H/RS cells has been hampered by their low frequency and fragility. Here, we describe the isolation of viable H/RS cells from HD affected tissues by high gradient magnetic cell sorting (MACS) according to expression of CD30. The cells were enriched to a purity of up to 50%. H/RS cells were distinguished from other CD30(+) cells by the expression of CD15, their size and granularity. No CD30/CD15 double-positive cells could be enriched from a lymph node affected by the lymphocyte predominant subtype of HD, activated lymph nodes or peripheral blood of healthy donors. For two cases of HD individual MACS-purified H/RS cells and H/RS cells micromanipulated from tissue sections of the same lymphoma specimens were analyzed for Ig gene rearrangements. In both cases, identical V gene rearrangements were amplified from both sources of H/RS cells, showing that H/RS cells were successfully enriched. Moreover, the finding that in both cases no additional Ig gene rearrangements other than the ones identified in the H/RS cells micromanipulated from tissue sections were amplified from the MACS-purified H/RS cells further supports the monoclonality of these cells throughout the affected lymph nodes. The isolation of viable H/RS cells ex vivo is prerequisite for a direct study of gene expression by those cells and of their interaction with cells in their vicinity.
霍奇金淋巴瘤（HD）的特征是在大量非恶性细胞中存在少量恶性霍奇金和里德-斯腾伯格（H/RS）细胞。H/RS细胞的分析因其低频率和脆弱性而受到阻碍。在此，我们描述了根据CD30的表达，通过高梯度磁性细胞分选（MACS）从HD受累组织中分离活的H/RS细胞。细胞富集至纯度高达50%。H/RS细胞通过CD15的表达、其大小和颗粒度与其他CD30(+)细胞区分开来。无法从HD淋巴细胞为主亚型受累的淋巴结、活化淋巴结或健康供体的外周血中富集到CD30/CD15双阳性细胞。对于两例HD，对单个MACS纯化的H/RS细胞以及从同一淋巴瘤标本的组织切片中显微操作得到的H/RS细胞进行了Ig基因重排分析。在这两例中，从两种来源的H/RS细胞中均扩增出相同的V基因重排，表明H/RS细胞成功富集。此外，在这两例中，除了从组织切片中显微操作得到的H/RS细胞中鉴定出的Ig基因重排外，从MACS纯化的H/RS细胞中未扩增出其他额外的Ig基因重排，这一发现进一步支持了这些细胞在整个受累淋巴结中的单克隆性。体外分离活的H/RS细胞是直接研究这些细胞的基因表达及其与周围细胞相互作用的前提条件。
