Martinez, Mark

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.

Abstract Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (“Mass-Fix”) has replaced serum immunofixation at our institution for detection of monoclonal immunoglobulins. One advantage of Mass-Fix is the ability to differentiate pathogenic endogenous monoclonal antibodies from therapeutic monoclonal antibodies (t-mabs), which are often the first-line treatment for multiple myeloma, by reviewing the light chain signature mass. The objective of this study was to describe the distribution of t-mabs and other key characteristics of clinical serum samples analyzed by Mass-Fix in 2022. Comprehensive results for each sample were extracted retrospectively from the laboratory information system. Of 66,710 total analyzed, 30,701 (46%) samples were reported as “positive” (i.e., contains a monoclonal protein), given the tertiary nature of our institution and reference laboratory services. The positive samples were further subdivided by t-mab (detected by Mass-Fix), with a total of 7,769 (25.3%) t-mabs reported. The top five IgG kappa t-mabs reported include daratumumab (n=6,537), elotuzumab or REGN5458/5459 (n=499), isatuximab (n=348), and rituximab (n=102). Rare observations included monoclonal proteins consistent with tixagevimab/ cilgavimab, for COVID-19 infection (n=7), obinutuzumab, an anti-CD20 t-mab for multiple sclerosis (n=1) and evolocumab, an IgG lambda anti-PCSK9 inhibitor (n=6). Of the 7,769 samples, the t-mab was the only reported clone in 47.7% of cases and was found along with the original disease-associated clone in remaining cases. When the original disease clone was present, it was an IgG kappa isotype 31% of the time and could have confounded the definition of complete therapy response for patients. 69% were non-IgG kappa original clones. Mass-Fix test orders have a multiple-choice prompt to provide any current therapy to aid in the patient’s result interpretation. For those samples where a t-mab was reported, the answer to this question regarding administered therapy was often discrepant from the report: only 38.5% of daratumumab-positive samples had an affirmation of daratumumab administration. The remaining 61.5% of daratumumab-positive samples reported an assortment of therapies, including bone marrow transplant, other t-mabs, no active therapy, or the field was left blank, highlighting poor compliance. Additionally, 154/36,009 samples where the provider affirmed daratumumab administration were reported as negative (i.e., no monoclonal protein observed on Mass-Fix). Often, the send-out laboratory does not have medical record access where treatment information is detailed. In conclusion, we reported the presence of a t-mab in 11.6% of all samples tested with Mass-Fix, suggesting that t-mabs have entered the clinical practice to stay, and accurately determining their presence in the challenging environment of poor compliance to prompt questions and evolving treatment regimens is something that cannot be ignored by clinical laboratories. Mass-Fix allows detection of a t-mab in the same routine run as other testing, without need of a second immunofixation to rule out the IgG kappa finding is not a t-mab.