MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins

Background: Plasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M-protein). Some patients have more than one identifiable M-protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M-proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M-protein, (2) an M-protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M-protein with light chain glycosylation, or (4) two distinct biclonal M-proteins. Methods: Patient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or Kappa and lambda light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgG Kappa M-proteins from t-mAbs. Mass differences between peaks were calculated to identify Nglycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples. Results: Eighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a tmAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated. Conclusions: We demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M-proteins is useful for monitoring of patients with PCDs.