显微切割的反应性淋巴组织增生中克隆性B细胞的检测：免疫球蛋白重链基因重排PCR分析中可能存在的诊断陷阱
Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement.
作者信息
Zhou X G, Sandvej K, Gregersen N, Hamilton-Dutoit S J
机构信息
Laboratory of Immunopathology, Institute of Pathology, Aarhus University Hospital, Denmark.
出版信息
Mol Pathol. 1999 Apr;52(2):104-10. doi: 10.1136/mp.52.2.104.
AIMS
To evaluate the specificity of standard and fluorescence based (Genescan) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations.
METHODS
PCR IgH gene rearrangement analysis of whole sections and microdissected fragments (n = 62) from paraffin wax embedded reactive lymph nodes (n = 6) and tonsils (n = 3). Amplificant analysis used both standard methods and automated high resolution fluorescence based quantification and size determination using GENESCAN software.
RESULTS
Whole tissue sections were consistently polyclonal in control experiments. IgH gene amplification was successful in 59 of 62 microdissected fragments; only two of 59 showed a polyclonal rearrangement pattern, the remainder being oligoclonal or monoclonal. Reanalysis was possible in 33 samples; six showed reproducible bands on gel analysis and satisfied accepted criteria for monoclonality. Use of high resolution gels with Genescan analysis improved sensitivity and band definition; however, three samples still appeared to be monoclonal.
CONCLUSIONS
These results confirm that PCR based IgH gene rearrangement analysis is a sensitive and specific method for demonstrating B cell clonality in whole paraffin wax embedded sections. However, oligoclonal and monoclonal rearrangement patterns are regularly encountered in small tissue fragments from otherwise unremarkable reactive lymphoproliferations, possibly because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present in more than one sample should be considered to be suggestive of neoplasia.
目的
评估标准聚合酶链反应（PCR）及基于荧光（基因扫描）的PCR免疫球蛋白重链（IgH）基因重排分析在完整及显微切割的石蜡包埋淋巴组织增殖切片中的特异性。
方法
对石蜡包埋的反应性淋巴结（n = 6）和扁桃体（n = 3）的全切片及显微切割片段（n = 62）进行PCR IgH基因重排分析。扩增产物分析采用标准方法以及使用GENESCAN软件进行的基于自动化高分辨率荧光的定量和大小测定。
结果
在对照实验中，全组织切片始终为多克隆性。62个显微切割片段中有59个IgH基因扩增成功；59个中只有2个显示多克隆重排模式，其余为寡克隆或单克隆。33个样本可进行重新分析；6个在凝胶分析中显示出可重复的条带，并符合单克隆性的公认标准。使用高分辨率凝胶和基因扫描分析提高了灵敏度和条带清晰度；然而，仍有3个样本似乎为单克隆性。
结论
这些结果证实，基于PCR的IgH基因重排分析是一种在完整石蜡包埋切片中显示B细胞克隆性的灵敏且特异的方法。然而，在原本无明显异常的反应性淋巴组织增殖的小组织片段中经常遇到寡克隆和单克隆重排模式，这可能是由于局部B细胞克隆的优先启动或检测所致。来自小的、显微切割的或淋巴细胞稀少样本的克隆分析数据必须进行严格评估。建议至少在两个组织标本上平行进行分析。只有在多个样本中出现的可重复条带才应被视为提示肿瘤形成。
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