Protein Electrophoresis Serum

Serum protein electrophoresis (SPEP) is ordered as a followup to other abnormal laboratory tests such as total protein, elevated urine protein, elevated calcium or an abnormal complete blood count. SPEP is most commonly ordered for patients with a suspected plasma cell malignancy such as multiple myeloma, Waldenstrom macroglobulinemia and primary amyloidosis. Signs and symptoms suggestive of the presence of a plasma cell disorder include:

• Unexplained anemia, back pain, weakness, or fatigue
• Osteopenia, osteolytic lesions, or spontaneous fractures
• Renal insufficiency with a nondiagnostic urine sediment
• Heavy proteinuria in a patient over age 40 years
• Hypercalcemia
• Hypergammaglobulinemia
• Immunoglobulin deficiency
• Bence Jones proteinuria
• Unexplained peripheral neuropathy
• Recurrent infections
• Elevated erythrocyte sedimentation rate or serum viscosity

Serum proteins differ in their size, shape, and electric charge and can be separated into 5 fractions by electrophoresis. Classically, separation has been performed on agarose gels. Proteins are visualized by staining with acid blue, and the intensity of staining is quantitated by densitometry. The concentration of each peak is determined by multiplying by its percentage by the serum total protein concentration.  

Capillary zone electrophoresis is a more automated and slightly more sensitive method of performing SPEP. Electro-osmotic flow is an all-important phenomenon in CE. If the buffer is above pH 2, the internal surface of the fused silica capillary is negatively charged due to exposed silanol ions. In an electric field, hydrated cations in the diffuse double-layer adjacent to the silica wall migrate towards the cathode, dragging solvent with them. This is termed electro-osmotic flow. The order of migration of proteins past the detector will reflect the balance between the electrophoretic and electro-osmotic forces within the capillary. By adjusting the pH of the buffer, electro-osmotic flow can, in principle, either enhance or oppose electrophoretic migration. In analysis of serum proteins, the pH used is markedly alkaline and the anodal electrophoretic migration is dominated by cathodal electro-endosmosis. Peptide bonds are detected by a spectrophotometer using wavelengths in the far UV spectrum, avoiding the need for protein staining. 

The band that migrates fastest toward the anode is albumin followed by alpha 1-globulin, alpha 2-globulin, beta globulin, and gamma globulins. The major proteins comprising each of the  5 fractions are:

• Albumin, which represents almost two-thirds of the total serum protein
• Alpha-1, composed primarily of alpha-1-antitrypsin (A1AT), orsomucoid
• Alpha-2, composed primarily of alpha-2-macroglobulin, haptoglobin, ceruloplasmin
• Beta-1 composed primarily of transferrin 
• Beta-2, beta lipoprotein and complement C3, IgA, IgM
• Gamma, composed primarily of immunoglobulins (Ig)

Protein concentrations may be altered as a result of different disease states. The concentration of these fractions and the electrophoretic pattern may be characteristic of diseases such as monoclonal gammopathies, A1AT deficiency disease, nephrotic syndrome, and inflammatory processes associated with infection, liver disease, and autoimmune diseases.

The most commonly recognized electrophoretic patterns are acute inflammation, alpha-1 antitrypsin deficiency, chronic inflammation, cirrhosis, hypoalbuminemia, hypogammaglobulinemia, monoclonal gammopathy, polyclonal gammopathy and protein losing disorder. 

Some valuable tips for interpreting SPEP patterns are:

• Bisalbuminemia is most commonly seen in Native American individuals
• Bilirubin, heparin & antibiotic binding can cause slurring of albumin band
• Hemolysis causes decreased alpha-2 band (haptoglobin) and appearance of hemoglobin band between alpha-2 and beta-1 regions
• C3 is labile & decreases with storage; results in much variation in beta-2 region
• Fibrinogen migrates between beta-2 and gamma regions (close to application point) and is present in plasma or heparin contaminated specimens
• CRP migrates in the gamma region and resembles a monoclonal band; present in acute inflammation
• Immune complexes may appear as a monoclonal band at the application point because they precipitate and do not migrate
• Oligoclonal bands with hypergammaglobulinemia & possibly beta-gamma bridging may be seen in patients responding to antigenic stimulation resulting from viral & bacterial infections, vaccines, autoimmune diseases, and angioimmunoblastic lymphadenopathy.  
• Oligoclonal bands with decreased IgG level is seen in CLL, after heart and bone marrow transplants, in common variable immunodeficiency, and during immunosuppressive Rx.
• Kappa chains usually stain more strongly than lambda chains

Characteristic Findings of Monoclonal Gammopathies on SPEP are:

• Monoclonal band (M-spike) is often in the gamma globulin region and more rarely in the beta or alpha-2 regions. 
• A hypogammaglobulinemic SPE pattern may suggest light chain disease.
•  Monoclonal bands in the beta region may indicate light chain disease, amyloidosis, heavy chain disease, IgD and IgE monoclonal gammopathies.
• A monoclonal IgG or IgA of greater than 3 g/dL is consistent with multiple myeloma (MM).
• A monoclonal IgG or IgA of less than 3 g/dL may be consistent with monoclonal gammopathy of undetermined significance (MGUS), primary systemic amyloidosis, early or treated myeloma, as well as a number of other monoclonal gammopathies.
• A monoclonal IgM of greater than 3 g/dL is consistent with macroglobulinemia.
• The initial identification of an IgM band greater than 4 g/dL,, IgA band greater than 5 g/dL, or IgG M band greater than 6 g/dL respectively, should be followed by serum viscosity.
• Gamma heavy chain disease can produce a relatively broad band anywhere from the alpha-2 through the gamma region
• Broader width of monoclonal bands may be related to amount of protein applied to gel or heterogeneity of monoclonal protein due to glycosylation. IgA monoclonal bands are usually broader than IgG.

Patients suspected of having a monoclonal gammopathy may have normal serum SPE patterns. Approximately 11% of patients with MM have a completely normal serum SPE, with the monoclonal protein only identified by MALDI-TOF MS. Approximately 8% of MM patients have hypogammaglobulinemia without a quantifiable M-spike on SPE but identified by MALDI-TOF MS. Accordingly, a normal serum SPE does not rule out the disease and SPE should not be used to screen for the disorder. SMOGA / Monoclonal Gammopathy Screening, Serum which includes MALDI-TOF MS and serum free light chains, should be done to screen if the clinical suspicion is high.

Clues that a monoclonal band is unlikely to be due to a malignant clonal expansion include:

• Acute phase pattern is present along with monoclonal band
• Infection can produce transient monoclonal bands that evolve into an oligoclonal pattern
• All immunoglobulin classes are elevated along with monoclonal band
• Slightly abnormal kappa:lambda ratio
• Bence Jones protein is absent from urine

After the initial identification of an M-spike, quantitation of the M-spike on follow-up SPE can be used to monitor the monoclonal gammopathy. However, if the monoclonal protein falls within the beta region (most commonly an IgA or an IgM) quantitative immunoglobulin levels may be more a useful tool to follow the monoclonal protein level than SPE. A decrease or increase of the M-spike that is greater than 0.5 g/dL is considered a significant change.

Serum protein electrophoresis should be repeated in one year for asymptomatic patients with a monoclonal protein less than 1.5 g/dL and normal values of hemoglobin, calcium, and creatinine.  Electrophoresis should be repeated in two to three months if the monoclonal protein is between 1.5 and 2.5 g/dL.  Patients being treated for multiple myeloma, Waldenstrom’s macroglobulinemia or amyloidosis should be monitored at one to two month intervals.  

Reference ranges using the Helena SPIFE 4000 Split Beta SPE system are:

Specimen requirement is a red top tube of blood. 

References

O’Connell TX, et al, Understanding and Interpreting Serum Protein Electrophoresis, Amer Fam Physician, 2005;71(1):105-112. 

Jenkins, M, Serum and Urine Electrophoresis for Detection and Identification of Monoclonal Proteins, Clin Biochem Rev, 2009;30:119-122.

Genzen JR, et al, Screening and Diagnosis of Monoclonal Gammopathies: An International Survey of Laboratory Practice, Arch Pathol Lab Med, 2018;142:507-515.

Kyle RA. Sequence of testing for monoclonal gammopathies. Arch Pathol Lab Med, 1999;123:114-8.

McCudden CR, et al, Synoptic reporting for protein electrophoresis and immunofixation, Clin Biochem, 2018;51:21-28.

Booth RA, et al, Candidate recommendations for protein electrophoresis reporting from the Canadian Society of Clinical Chemists Monoclonal Gammopathy Working Group, Clin Biochem, 2018;51:10-20.