False-negative serum protein electrophoresis in a sample with an IgM monoclonal protein by semiautomated gel electrophoresis.

To the Editor: In a recent report, Keren et al. (1 ) described a case in which the Sebia semiautomated electrophoresis method failed to demonstrate proteinuria in a 50-fold concentrated urine sample despite the presence of 2 g/24 h total protein. The reason for this failure was attributed to the presence of debris associated with cellular elements that interfered with the wicking of the urine into the microporous membrane applicator. The manufacturer is aware of such problems and recommends centrifugation of turbid urine samples. In our laboratory, all urine samples are systematically centrifuged (10 min at 1124g), and to date, we have not encountered such a failure of the Sebia system. We did, however, analyze a serum sample for which agarose electrophoresis with the Hydrasys analyzer (Sebia, Inc.), using Hydragel 15 HR gels (Sebia), missed a large (12 g/L determined on a Beckman-Coulter Immage nephelometer) IgM monoclonal protein (2 ) (Fig. 1A). Similarly, immunofixation with the Sebia system using Hydragel 4 IF (Sebia) gels failed to detect the monoclonal protein (data not shown). The monoclonal protein was detected by capillary zone electrophoresis (Fig. 1B) and by manual agarose electrophoresis using the Beckman Paragon agarose electrophoresis system. Manual immunofixation (Paragon; BeckmanCoulter) was used to characterize the monoclonal protein (data not shown). We ascribed the failure of the Sebia semiautomated system to the use of plastic applicators to apply the serum and hypothesized that the IgM monoclonal protein remained adhered to the microporous membrane of the applicator and did not diffuse into the gel. Mercaptoethanol treatment [10 L of a 1:10 dilution of mercaptoethanol (Merck cat. no. 15433) added to 100 L of serum] of the sample to cleave the disulfide bonds in the IgM pentamer revealed the monoclonal protein with the semiautomated Hydrasys system (Fig. 1C). The company recommends treating viscous or turbid serum samples (particularly those containing cryoglobulin or cryogel) with Fluidil (prod. no. 4587; Sebia). Such treatment (25 L of Fluidil added to 75 L of serum), however, did not resolve the problem. Sebia also recommends use of 10 g/L -mercaptoethanol in Fluidil for proteins that can polymerize, which would produce a “monoclonal fraction” in all immunofixed tracks. This, however, was not the case for the sample described. By contrast, the system failed to detect a monoclonal fraction. We conclude that the applicator used with the semiautomated electrophoresis system from Sebia may give problems with some rare serum samples that contain an IgM monoclonal protein. Additional techniques to evaluate the presence of monoclonal proteins should therefore be available, such as nephelometry, capillary zone electrophoresis, and treatment with 2-mercaptoethanol.