Chemistry Case Study: Protein Bands in All Lanes of the Immunofixation Electrophoresis

Waldenstrom Macroglobulinemia (WM) is defined as lymphoplasmacytic lymphoma (LPL) with IgM paraprotein and bone marrow involvement. The IgM paraprotein is an important serum marker for WM diagnosis, symptom prediction, disease burden assessment, treatment decision and drug response evaluation. Serum protein electrophoresis (SPEP) in conjunction with immunofixation electrophoresis (IFE) are the routine laboratory tests for IgM paraprotein detection, quantitation and characterization. A monoclonal protein typically presents as a sharp band on SPEP and selective lanes of IFE, allowing characterization of the immunoglobulin heavy chain isotypes and light chain classes. In rare situations, a monoclonal band is seen on all immunofixation lanes, suggesting cryoglobulin and/or soluble immune complex. We encountered a recent case of WM with a strong demarcated band on all immunofixation lanes.

The patient is a 76-year-old Chinese man diagnosed as WM/LPL by bone marrow biopsy. Peripheral blood showed pancytopenia with rouleaux formation. The serum IgM was up to 6900 mg/dL. Serum viscosity was increased up to 3.1 cP (normal range 1.5-1.9 cP). Serum rheumatoid factor was negative (<10 IU/ml). Serum protein electrophoresis (SPEP) on Sebia Hydrasys 2 showed a wide smearing pattern (Fig 1A). Serum protein immunofixation electrophoresis (IFE) showed a monoclonal band on all lanes with equal intensity, preventing isotype identification (Fig 1B). This pattern is generally believed to be due to cryoglobulin and/or polymerization of monoclonal proteins, similar to rheumatoid factor activity. Urine electrophoresis was consistent with an overflow pattern and urine immunofixation showed monoclonal free lambda light chain.

Previously it was demonstrated that cryogolublin dissolution was achieved by pre-treatment of serum samples with Fluidil. The IgM polymer can be disrupted by adding reducing agents such as beta-mercaptoethanol (bME) to disrupt the disulfide bonds (1-2). In our case, despite pretreatment with Fluidil and bME, no isotype resolution was achieved on serum IFE, prompting us to develop a novel method through the addition of sodium dodecyl sulfate (SDS) to the pretreatment process. Different combinations of reaction conditions were tested, including SDS concentration ranging from 0.01 to 1%, three different temperatures (37, 56 and 95 °C), three different concentrations of bME (1%, 2% and 4%) and three different serum volume (25 µL, 50 µL and 75 µL). Optimal isotype resolution was achieved using 0.1% SDS/0.25%bME/Fluidil incubated at 56°C for 30 mins (Fig 1C).

References

1. Attaelmannan M, Levinson SS. Understanding and identifying monoclonal gammopathies. Clin Chem. 2000 Aug; 46(8 Pt 2):1230-8.
2. Yusra Othman. Protein Bands in All Lanes of the Immunofixation Electrophoresis Pattern of Serum From a 50-Year-Old Saudi Woman. Lab Med (2006) 37 (3): 152-154.

-Huifei Liu, MD, PhD. Former PGY4 resident in the Department of Pathology and Genomic Medicine, Houston Methodist Hospital. She currently serves as the associate medical director at Hematologics, Inc., Seattle, WA.  

-Xin Yi, PhD, DABCC, FACB, is a board-certified clinical chemist, currently serving as the Co-director of Clinical Chemistry at Houston Methodist Hospital in Houston, TX and an Assistant Professor of Clinical Pathology and Laboratory Medicine at Weill Cornell Medical College.