The clonal product, when present, was excised from gel, purified by the MinElute gel-extraction method (Qiagen), and sequenced with heavy-chain reverse primer with the use of BigDye Terminator, version 3.1, on a ABI 3730 Genetic analyzer (Applied Biosystems). Subcloning was used for clonal cases occurring on a polyclonal background with the use of a TOPO TA cloning kit (Invitrogen). The PCR products were ligated into the vector and transformed into Escherichia coli cells. Eight colonies were randomly selected for sequencing, as described above. Nucleotide sequences were analyzed with the use of the IMGT database and tools30 and aligned to the closest match with the germ-line IGHV segment. Sequences with a germ-line identity of less than 98% were considered to be mutated, whereas those with a germ-line identity of 98% or more were considered to be unmutated.31,32
