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Inaccurate Quantitation of IgM Monoclonal Proteins: A cause for concern
Author(s) -
Holmes Earle W.,
Bahar Burak,
Whisler Barbara M.,
Gryzbac Margie,
Kahn Stephen E.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.696.3
Subject(s) - chemistry , albumin , chromatography , monoclonal , monoclonal antibody , microbiology and biotechnology , medicine , immunology , antibody , biochemistry , biology
The detection and quantification of monoclonal proteins (MCP) play an essential role in the diagnosis and management of plasma cell dyscrasias. As a follow‐up to an unusual case in which our SPEP method severely underestimated the size of an IgM monoclonal protein in a patient with a provisional diagnosis of amyloidosis, we investigated the linearity of the Sebia Hydrasys, Hydragel beta 1–2 method ( Sebia, Inc. Norcross, GA 30093) for the recovery of IgM(n=19) and IgG(n=9) MCP using the manufacturer's routine protocol both before and after pre‐dilution of the serum with saline (SAL) or a chaotropic diluent containing a mild reducing agent(DPD). MCP concentrations in SPEP patterns from amido black‐stained gels were quantitated by the routine Sebia protocol, and the recoveries of the albumin and MCP bands were directly measured as total RBG intensities by image analysis(IA) using Image Studio Lite, 4.0 (Li‐Cor, Lincoln NE 68504). Results are presented as mean (min to max). IA showed that the mean recoveries of the albumin and MCP bands of diluted samples containing IgM MCP were increased by factors of 3.0‐fold (1.6‐ to 10.6) and 4.1‐fold (1.5 to 24.6), respectively, over those measured in the undiluted samples. Moreover, the MCP/albumin recovery ratio was increased by 1.2‐fold (0.7 to 2.4) compared to the undiluted serum. This ratio was significantly increased compared to the ratio of 0.95‐fold (0.8 to 1.1) observed for the specimens that contained IgG MCP (p<0.001, KW). These results showed that the routine Sebia method, which specifies the analysis of undiluted serum, frequently under‐estimated the concentration of IgM MCP, possibly owing to flaws in the sample application and/or the protein staining steps of the procedure. Pre‐dilution with SAL prior to analysis increased IgM MCP concentrations by a mean of 9.4 % (0 to 22.0). Pre‐dilution with DPD provided similar increases except in one case in which the concentrations of two, relatively small, IgM MCP were increased by 22 and 52%. In addition to an under‐recovery of IgM MCP by SPEP, we found that band size estimates based on the quantitation of the total IgM concentration by rate nephelometry (Immage 800, Beckman Coulter Inc., Brea CA 92821) were 1.3‐ to 2.7‐fold greater than those measured by SPEP. Both of the major methods used for estimating the size of MCP appear to have analytical limitations that compromise their accuracy. While either method might be adequate for monitoring changes in tumor burden over time, the discrepancies we noted may be a source of confusion and error in scenarios in which fixed clinical decision thresholds based on MCP concentration are used as criteria for diagnosis, disease progression, and response to treatment.