Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell nan Hodgkin's lymphoma. A comparison between different strategies

Background and Objective. The main difficulty of PCR-based clonality studie s for B-cell lymphoproliferative disorders (B-LPD) is discrimination betwee n monoclonal and polyclonal PCR products, especially when there is a high b ackground of polyclonal B cells in the tumor sample. Actually, PCR-based me thods for clonality assessment require additional analysis of the PCR produ cts in order to discern between monoclonal and polyclonal samples. Heterodu plex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. Design and Methods. We studied the sensitivity and specificity of heterodup lex PCR analysis for monoclonal detection in samples from 90 B-cell non-Hod gkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B -cell disorders (negative controls). Furthermore, in 42 B-NHL and in the sa me 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO3) staining as well as agarose vs, polyacrylamide gel electrophoresis (PAGE). Results. Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex P CR analysis using PAGE and AgNO3 staining. Moreover, no polyclonal sample s howed a monoclonal PCR product. By contrast, false positive results were ob tained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/ 28 and 8/28 with EtBr and AgNO3 staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 i n PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex an alysis. Interpretation and Conclusions. We conclude that AgNO3 stained PAGE after h eteroduplex 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. (C) 1999, Ferr ata Storti Foundation.