d'Amore F, Stribley J A, Ohno T, Wu G, Wickert R S, Delabie J, Hinrichs S H, Chan W C
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-3135, USA.
Lab Invest. 1997 Feb;76(2):219-24.
Molecular analysis of isolated single cells is a powerful tool for analyzing heterogeneity within a population of cells and for clarifying issues of cell origin and clonality. Current techniques are limited by the availability of suitable fresh tissue. To broaden the applicability of molecular techniques at single-cell level, we have developed an approach that uses routinely processed archival tissue. Immunoglobulin heavy chain (IgH) gene rearrangement was analyzed in large tumor cells from four cases of diffuse large cell B-non-Hodgkin's lymphoma and in small reactive T and B lymphocytes from three cases of lymphocytic predominance Hodgkin's disease. One case of Epstein-Barr virus (EBV)-encoded RNA (EBER)-positive angiocentric pulmonary T-cell lymphoma was assayed for the presence of the BamHI-W multiple-copy fragment of the EBV genome. T- and B-lymphoid cells were immunostained with anti-CD3 and CD20, respectively. The tissue sections from the EBER-positive T-cell lymphoma were stained by nonisotopic in situ hybridization. Single cells were mobilized after proteolytic treatment under an inverted microscope using a hydraulic micromanipulator at a magnification of 400 x. Isolated cells were aspirated into a micropipette fixed to a second micromanipulator and transferred into a PCR tube. The IgH complementarity determining region (CDR)3 was successfully amplified in 17 of 52 (33%) small B-lymphocytes from lymphocytic predominance Hodgkin's disease using a previously reported semi-nested PCR method, and the products from each case differed in size as expected of a polyclonal population. None of the 49 small T lymphocytes demonstrated any amplifiable IgH CDR3 products, indicating no significant cellular contamination. The IgH CDR3 sequence analysis of the PCR products indicated a clonal relationship among harvested cells. In the T-cell lymphoma case, the harvested EBER-positive cells were amplifiable for the multiple-copy fragment BamHI-W of the EBV genome. Our study indicates that single-cell analysis can be performed on paraffin-embedded archival tissue after being subjected to immunoperoxidase and in situ hybridization procedures.
对分离出的单个细胞进行分子分析，是剖析细胞群体异质性以及阐明细胞起源和克隆性问题的有力工具。当前技术受限于合适新鲜组织的可获取性。为拓宽分子技术在单细胞水平的适用性，我们开发了一种利用常规处理存档组织的方法。对4例弥漫性大细胞B细胞非霍奇金淋巴瘤的大肿瘤细胞以及3例淋巴细胞为主型霍奇金病的小反应性T和B淋巴细胞进行了免疫球蛋白重链（IgH）基因重排分析。对1例爱泼斯坦-巴尔病毒（EBV）编码RNA（EBER）阳性的血管中心性肺T细胞淋巴瘤检测了EBV基因组的BamHI-W多拷贝片段。T和B淋巴细胞分别用抗CD3和CD20进行免疫染色。EBER阳性T细胞淋巴瘤的组织切片通过非同位素原位杂交染色。在倒置显微镜下，使用液压显微操作器在400倍放大倍数下进行蛋白水解处理后分离单个细胞。将分离出的细胞吸入固定在第二个显微操作器上的微量移液器中，并转移到PCR管中。使用先前报道的半巢式PCR方法，在52个来自淋巴细胞为主型霍奇金病的小B淋巴细胞中有17个（33%）成功扩增出IgH互补决定区（CDR）3，并且每个病例的产物大小不同，符合多克隆群体的预期。49个小T淋巴细胞均未显示出任何可扩增的IgH CDR3产物，表明无明显细胞污染。PCR产物的IgH CDR3序列分析表明收获的细胞之间存在克隆关系。在T细胞淋巴瘤病例中，收获的EBER阳性细胞可扩增出EBV基因组的多拷贝片段BamHI-W。我们的研究表明，石蜡包埋的存档组织在经过免疫过氧化物酶和原位杂交程序后可进行单细胞分析。
