González M, González D, López-Pérez R, García-Sanz R, Chillón M C, Balanzategui A, Mateos M V, Alaejos I, Langerak A W, Orfão A, Van Dongen J J, San Miguel J F
Service of Hematology, University Hospital of Salamanca, Paseo de San Vicente, 58-182, Salamanca, 37007, Spain.
Haematologica. 1999 Sep;84(9):779-84.
The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment.
We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE).
Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis.
We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.
基于聚合酶链反应（PCR）的B细胞淋巴增殖性疾病（B-LPD）克隆性研究的主要困难在于区分单克隆和多克隆PCR产物，尤其是当肿瘤样本中多克隆B细胞背景较高时。实际上，基于PCR的克隆性评估方法需要对PCR产物进行额外分析，以便区分单克隆和多克隆样本。异源双链分析是一种有吸引力的方法，因为它易于操作，且无需使用放射性底物或昂贵设备。
我们研究了异源双链PCR分析在90例B细胞非霍奇金淋巴瘤（B-NHL）患者样本及28例无肿瘤性B细胞疾病个体（阴性对照）中检测单克隆的敏感性和特异性。此外，在42例B-NHL患者及相同的28例阴性对照中，我们比较了异源双链分析与经典PCR技术。我们还比较了溴化乙锭（EtBr）与硝酸银（AgNO₃）染色以及琼脂糖与聚丙烯酰胺凝胶电泳（PAGE）。
使用位于VH（FR3和FR2）及JH的两对共有引物，91%的B-NHL样本在采用PAGE和AgNO₃染色的异源双链PCR分析后显示出单克隆产物。此外，没有多克隆样本显示出单克隆PCR产物。相比之下，使用琼脂糖（5/28）和未进行异源双链分析的PAGE时会得到假阳性结果：分别使用EtBr和AgNO₃染色时，假阳性结果分别为2/28和8/28。此外，假阴性结果仅出现在EtBr染色时：琼脂糖中为13/42，未进行异源双链分析的PAGE中为4/42，异源双链分析后的PAGE中为7/42。
我们得出结论，异源双链分析后用AgNO₃染色的PAGE是检测B-NHL样本中单克隆重排的最合适策略，因为它不会产生假阳性结果，且假阴性结果的风险非常低。
