IMPROVED PCR METHOD FOR DETECTING MONOCLONAL IMMUNOGLOBULIN HEAVY-CHAIN REARRANGEMENT IN B-CELL NEOPLASMS

Aims: To develop a simple, optimised, polymerase chain reaction (PCR) based method for detecting the rearranged immunoglobulin heavy chain (IgH). Methods: Using as primers oligonucleotides (Fr2A, Fr2B) homologous to the conserved sequences to the framework II region and the joining (J(H)) region, 25 patients with B cell lymphoproliferative disorders, previously characterised by Southern blotting, and three patients with light chain myeloma were studied. Results: The PCR product from a polyclonal B cell population showed a broad band when analysed on a 3% agarose gel; DNA from B cell lines and B lymphoproliferative disorders showed a discrete band. Specificity of the amplification was confirmed by cloning and sequencing the amplified product as well as by Southern blotting with an internal probe homologous to the framework 3 region. Primers Fr2A and Fr2B detected monoclonality in three patients with light chain myeloma, while primers directed against the FrIII region showed a polyclonal response. Conclusions: Deletions and extensive somatic mutations within the FrIII region may give false negative results with primers homologous to the region. A PCR using the method described, with a repertoire of primers homologous to the FrII and FrIII regions, will therefore increase the frequency of detection of monoclonality.