Mass Spectrometric Exploration of Biclonal Gammopathy: Insights from a Diagnostic Laboratory

Biclonal gammopathy is observed in approximately 1% of myeloma cases. Most current understanding of this condition is derived from incidental or anecdotal findings based on serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). The widespread use of therapeutic monoclonal antibodies (tmAbs), such as daratumumab, has introduced additional complexity, as these agents may appear as monoclonal bands on IFE, potentially interfering with accurate diagnosis. This study utilizes the enhanced sensitivity and molecular specificity of liquid chromatography-triple quadrupole mass spectrometry (LC-TQMS) to characterize biclonal patterns in patient sera. Over a 3.3-year period (January 2022 to April 2025), 124 cases initially reported as biclonal by IFE were reviewed, of which 85 samples were selected for LC-TQMS analysis following immunoglobulin enrichment using camelid antibodies targeting immunoglobulin isotypes and light chains. Released light chains, following reduction, were separated by liquid chromatography, and their molecular masses determined by TQMS. Among the isotype combinations, IgG-IgA was the most frequent (34.3%), followed by IgG-IgG (31.4%), while IgG-IgM and IgM-IgM combinations were each observed in 2.9% of cases. Regarding light chain patterns, κ-λ combinations were observed in 40% of cases, λ-λ in 25.7%, and κ-κ in only 5.7%, the latter likely reflecting tmAb interference. LC-TQMS analysis revealed biclonal patterns in 31.8%, monoclonal patterns in 52.9%, and tmAb interference in 9.4%. LC-TQMS demonstrated robust capability in resolving biclonality, including cases with or without coexistent free light chains, and in distinguishing analytical artifacts due to tmAb or conformational changes in immunoglobulin light chains that may mimic biclonal patterns on IFE. These findings underscore the importance of incorporating treatment history into IFE interpretation and support the implementation of LC-TQMS as an orthogonal method for resolving complex gammopathy profiles. A workflow is proposed to aid in accurate interpretation of biclonal cases.