Identification and quantitation of monoclonal proteins in polyclonal hypergammaglobulinemia background: a retrospective study

Abstract Background Hypergammaglobulinemia poses a challenge for identifying and quantitating monoclonal proteins (M-protein). The Increased polyclonal immunoglobulin background can lead to imprecise estimation of the M-protein when quantitated by serum protein electrophoresis (SPEP). This is especially true using the perpendicular drop method of peak integration. Hypergammaglobulinemia also makes it difficult to detect small M-proteins on immunofixation electrophoresis. Overestimation of M-protein concentration can lead to unnecessary follow-ups while missing small M-proteins can mask the underlying diagnosis for patients. In this study, we retrospectively evaluated the prevalence and concentration of M-protein within polyclonal hypergamma backgrounds using immunocapture matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MASS-FIX). Methods We retrospectively analyzed 47061 unique patients tested for SPEP and MASS-FIX in 2022 at Mayo Clinic. If a patient underwent multiple tests during this period, only the first result was included in the analysis. Polyclonal hypergammaglobulinemia was defined as a gamma fraction >1.7g/dL by SPEP. MASS-FIX was used to identify and isotype M-proteins. MASS-FIX was then combined with total immunoglobin quantitation (IgG, IgA or IgM) using nephelometry to quantify concentration of the M- protein using mathematical modeling (method currently under validation). Results Of the 47,061 unique patients, 24,796 were normal by SPEP (53%). M-spikes were present in 5,808 cases (12%). Hypogammaglobulinemia (gamma fraction < 0.5g/dL) in 2897 cases (6%). Small non-quantifiable M-proteins by SPEP were present in 8363 cases (18%). [JC2] There were 4222 hypergamma cases in the cohort (9%), without a quantitative M-spike on SPEP. Of these, 16.1% (680/4222) patients had an M-protein by MASS-FIX. When polyclonal hypergamma was the sole SPEP abnormality, 5.5% were MASS-FIX positive. However, when a small abnormality pattern was observed within the polyclonal hypergamma background on SPEP, 50% of patients were MASS-FIX positive. The most common isotype was IgG (27.2%), followed by IgM (10.2%), IgA (8.1%), and biclonal (3.7%), which is similar to overall isotype distribution in other backgrounds. Using quantitative MASS-FIX, we identified that 95% MASS-FIX positive small monoclonal proteins in polyclonal hypergamma are < 1 g/dL, which is the recommended limited of quantitation for M-protein using SPEP in setting of hypergammaglobulinemia. Conclusion Majority of polyclonal hypergammaglobulinemia cases (83.9%) do not have a monoclonal protein. MASS-FIX allows quantitation of small monoclonal proteins below the SPEP limit of quantitation, which can enhance our understanding of conditions which present with small M-proteins in a polyclonal hypergammaglobulinemia background. Small visual abnormalities within polyclonal hypergamma background on SPEP should be followed-up with immunofixation or MASS-FIX to confirm the presence or absence of M- proteins.