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Heteroduplex analysis in discrimination between monoclonal and polyclonal PCR products

Background. PCR-based clonality methods are crucial for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes in case of suspected Non- Hodgkin΄s lymphoma (NHLs), but require additional analysis of PCR products for discrimination between monoclonal and polyclonal samples. Although the test manufacturers suggested agarose TBE or polyacrylamide TBE gels detection as one way of detection, according to European BIOMED-2 collaborative study only two techniques are recomended: heteroduplex analysis or GeneScanning. After unexpected PCR amplification gel detection results, we performed PCR amplification followed by heteroduplex analysis. Aim. To compare PCR amplification followed by heteroduplex analysis gel detection and PCR amplification gel detection. Methods. DNA samples taken from 15 patients with suspected NHLs are amplified with multiplex master mixes containing consensus DNA primers that target different regions within Ig/TCR genes (IgH and IdentiClone TCRB Gene Clonality Assay, Ipsogen). PCR amplification followed by heteroduplex analysis gel detection and PCR amplification gel detection were analysed in parallel. Clonal rearrangements were identified as single-sized PCR products inside the valid size range. Results. Final PHD diagnosis of NHL was found in 47% (7/15) of suspected NHLs whereas 40% were reactive (6/15). In 6 cases of 7 NHLs with PCR amplification gel detection clonal rearrangement was found while in only 2 of 7 NHLs clonal rearrangement after PCR amplification followed by heteroduplex analysis gel detection was found. Conclusion. PCR amplification followed by heteroduplex analysis gel detection in cases of suspected NHLs reduces false- positive PCR results and therefore increased the test specificity.

PCR-based clonality; Non- Hodgkin΄s lymphoma; heteroduplex analysis

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