Yakirevich Evgeny, Jackson Cynthia L, Meitner Patricia A, MacKenzie Dolores, Tavares Rose, Robinson-Bostom Leslie, DeLellis Ronald A, Resnick Murray B
Department of Pathology, Rhode Island Hospital, APC-12, 593 Eddy St., Providence, RI 02903, USA.
J Mol Diagn. 2007 Sep;9(4):490-7. doi: 10.2353/jmoldx.2007.070006. Epub 2007 Jul 9.
Identification of clonal lymphocytic populations by polymerase chain reaction may be difficult in cases with scant cellular infiltrates or those with a heterogeneous population of cells. Here, we assessed the diagnostic utility of laser capture microdissection (LCM) and high-resolution microcapillary electrophoresis in the analysis of clonality of small biopsy specimens. Clonality was determined in 24 cases: five reactive tonsils, five reactive lymph nodes, six inflammatory skin lesions, and eight T-cell lymphomas. CD3-positive T lymphocytes were captured by LCM from paraffinized immunohistochemically stained sections. Genomic DNA was analyzed for T-cell receptor-gamma gene rearrangement by polymerase chain reaction followed by high-resolution microcapillary electrophoresis with the DNA 500 LabChip and the Agilent Bioanalyzer. In the reactive specimens, T-cell receptor-gamma polymerase chain reaction revealed monoclonal bands when 10 to 1000 cells were captured. This pattern changed to polyclonal when higher numbers of cells were microdissected (2000 to 10,000 cells). In contrast, lymphoma cells were consistently monoclonal whether low or high numbers were microdissected. Microcapillary electrophoresis coupled with LCM facilitated clonality analysis in equivocal cases. In two of eight lymphoma cases, LCM revealed diagnostic monoclonal bands, whereas routine T-cell receptor-gamma assessment of whole tissue sections with 10% polyacrylamide gel electrophoresis demonstrated only minor clonal bands. We conclude that clonality determined by LCM is cell number-dependent. Biopsy specimens containing low numbers of reactive polyclonal T cells may produce pseudomonoclonal bands and therefore should be interpreted with great caution.
对于细胞浸润稀少或细胞群体异质性的病例，通过聚合酶链反应鉴定克隆性淋巴细胞群体可能会很困难。在此，我们评估了激光捕获显微切割（LCM）和高分辨率毛细管电泳在小活检标本克隆性分析中的诊断效用。对24例病例进行了克隆性测定：5例反应性扁桃体、5例反应性淋巴结、6例炎性皮肤病变和8例T细胞淋巴瘤。通过LCM从石蜡包埋的免疫组化染色切片中捕获CD3阳性T淋巴细胞。通过聚合酶链反应分析基因组DNA的T细胞受体γ基因重排，随后使用DNA 500 LabChip和安捷伦生物分析仪进行高分辨率毛细管电泳。在反应性标本中，当捕获10至1000个细胞时，T细胞受体γ聚合酶链反应显示单克隆条带。当显微切割的细胞数量较多（2000至10000个细胞）时，这种模式变为多克隆。相比之下，无论显微切割的细胞数量是多还是少，淋巴瘤细胞始终是单克隆的。毛细管电泳与LCM相结合有助于在疑难病例中进行克隆性分析。在8例淋巴瘤病例中的2例中，LCM显示出诊断性单克隆条带，而用10%聚丙烯酰胺凝胶电泳对整个组织切片进行常规T细胞受体γ评估仅显示出少量克隆条带。我们得出结论，LCM确定的克隆性取决于细胞数量。含有少量反应性多克隆T细胞的活检标本可能会产生假单克隆条带，因此在解释时应格外谨慎。
