Improved PCR method for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell neoplasms.

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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 (JH) 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.

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