Type II mixed cryoglobulinaemia as an oligo rather than a mono B-cell disorder: evidence from GeneScan and MALDI-TOF analyses

Objective. To identify and characterize rheumatoid factor (RF)-producing B-cells and cryoprecipitate immunoglobulin (Ig) M in hepatitis C virus (HCV)-positive patients. Methods. We purified and characterized, by peptide mass fingerprinting integrated with an NCBI IgBlast data bank search, the IgM component of cryoprecipitate and analysed the VDJ pattern of bone marrow B-cells by gene scan analysis of 17 HCV-positive patients with type II mixed-cryoglobulinaemia. Results. IgM purified from all of the patients presented an RF specificity. In three of these patients a high and predominant B-cell clone (>= 30%) was found in the bone marrow. B-cell-receptor sequences were determined and immunophenotyping of these clones was performed. Peptide masses originating after tryptic digestion of the B-cell-receptor combinatory regions and those originating by tryptic digestion of the cryoprecipitated IgM from the same patient were comparable. In the remaining patients an oligoclonal/polyclonality was found. However, in some of these patients we were able to find peptides that matched with the B-cell-receptor sequences of overexpanded B cells, indicating that, even in the absence of a clear monoclonal expansion, a fraction of total cryoprecipated IgM may derive from overexpanded B-cell clones found in patients' bone marrow. Conclusions. In the majority of mixed cryoglobulinaemia-HCV-positive patients, both in the serum and in B cells from the bone marrow, an oligoclonal pattern is the main molecular picture. When a monoclonal B-cell clone is found, its B-cell-receptor shows an antigen-binding fragment identical to that of cryoprecipitable RF-IgM. Phenotypically, B cells are CD20-positive but CD5-negative, suggesting that the B-1 B-cell subset is not likely to produce high-affinity IgM-RF molecules.