Lymphocyte predominance Hodgkin's disease: lineage and clonality determination using a single-cell assay.
Lymphocyte predominance Hodgkin's disease: lineage and clonality determination using a single-cell assay.
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淋巴细胞为主的霍奇金病：使用单细胞测定测定谱系和克隆性。
作者：
J. Delabie;A. Tierens;G. Wu;D. Weisenburger;W. Chan
淋巴细胞为主型霍奇金病 (LPHD) 是一种临床惰性病症。尽管有证据表明该疾病中假定的肿瘤细胞“L&H”细胞属于 B 细胞谱系，但有关这些细胞克隆性的数据存在矛盾。我们的研究旨在通过单细胞测定阐明 LPHD L&H 细胞的谱系和克隆性问题。研究了 4 例 LPHD 病例。为了克服获取新鲜组织的困难并能够研究代表性病例，开发了一种新方法，从档案福尔马林固定石蜡包埋的组织中获取 L&H 细胞的单细胞悬浮液。通过形态学和上皮膜抗原免疫染色来鉴定单个 L&H 细胞，使用微量移液管进行分离，并对 B 细胞克隆特异性的 Ig 重链 (IgH) 基因的互补性决定区 3 (CDR3) 进行聚合酶链式反应 (PCR) 扩增。 PCR产物通过聚丙烯酰胺凝胶电泳进行大小分级，并直接对代表性产物进行测序。还从组织中分离出单个T细胞和小B细胞，并分别用作阴性和阳性对照。在所有四种 LPHD 病例中，单个 L&H 细胞的 IgH CDR3 都可以被扩增。在每种情况下，发现单个 L&H 细胞的 IgH CDR3 具有不同的长度或不同的序列。因此，我们的结果为 L&H 细胞的 B 细胞起源和 LPHD 的多克隆性质提供了强有力的证据。
Lymphocyte predominance Hodgkin's disease (LPHD) is a clinically indolent condition. Although there is evidence that the putative neoplastic cell in this disease, the "L&H" cell, is of B-cell lineage, there is conflicting data concerning the clonality of these cells. Our study was aimed at clarifying the issue of lineage and clonality of the L&H cells of LPHD using a single-cell assay. Four cases of LPHD were studied. To circumvent the difficulties of obtaining fresh tissue and to be able to study representative cases, a new method was developed to obtain single-cell suspensions of L&H cells from archival formalin-fixed paraffin-embedded tissue. Single L&H cells were identified by morphology and immunostaining for epithelial membrane antigen, isolated using a micropipette, and subjected to polymerase chain reaction (PCR) amplification of the complematarity determining region 3 (CDR3) of the Ig heavy chain (IgH) gene, which is B-cell clone-specific. The PCR products were size-fractionated by polyacrylamide gel electrophoresis and representative products were directly sequenced. Single T cells and small B cells were also isolated from the tissues and used as negative and positive controls, respectively. In all four cases of LPHD, the IgH CDR3 of single L&H cells could be amplified. Within each case, the IgH CDR3 of single L&H cells was found to be of different length or of different sequence. Therefore, our results provide strong evidence for the B-cell origin of the L&H cells and the polyclonal nature of LPHD.
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作者：
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DOI：
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期刊：
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影响因子：
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DOI：
--
发表时间：
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期刊：
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影响因子：
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DOI：
10.1073/pnas.86.13.5123
发表时间：
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影响因子：
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