Serum protein electrophoresis (SPEP) is an easy, inexpensive method of separating proteins based on their net charge, size, and shape. The 2 major types of protein present in the serum are albumin and the globulin proteins. Albumin is the major protein component of serum and represents the largest peak that lies closest to the positive electrode.  Globulins comprise a much smaller fraction of the total serum protein but represent the primary focus of interpretation of serum protein electrophoresis. Five globulin categories are represented: alpha-1, alpha-2, beta-1, beta-2, and gamma, with the gamma fraction being closest to the negative electrode.
The reference range for serum protein electrophoresis is as follows:
Albumin: 3.3-5.7 g/dL
a- 1: 0.1-0.4 g/dL
a- 2: 0.3-0.9 g/dL
b- 2: 0.7-1.5 g/dL
gamma: 0.5-1.4 g/dL
The reference range may vary depending on the laboratory performing the test.
Various disease states or conditions alter the pattern of proteins in electrophoresis (see Table 1 below).
Table 1. Serum Protein Fractions and Conditions Associated with an Increased or Decreased Level (Open Table in a new window)
|Serum Protein Fraction||Increased||Decreased|
|Albumin||Severe dehydration||Malnutrition, cachexia, liver disease, nephrotic syndrome, protein-losing enteropathies, severe burns|
|Alpha-1||Inflammatory states, pregnancy||Alpha-1 antitrypsin deficiency|
|Alpha-2||Inflammatory states, nephrotic syndrome, oral contraceptive use, steroid use, hyperthyroidism||Hemolysis, liver disease|
|Beta||Hyperlipidemia, iron-deficiency anemia||Hypo-B-lipoproteinemia, malnutrition|
|Gamma||Polyclonal and Monoclonal Gammopathies||Agammaglobulinemia, hypogammaglobulinemia|
A dense narrow band that is composed of a single class of immunoglobulins secreted by an abnormally expanded clone of plasma cells is known as M-protein (paraprotein, monoclonal protein or M-component).  An M-protein usually presents as a single narrow peak, resembling a "church spire," in the gamma, beta, or alpha-2 region of the densitometer tracing, or as a dense, discrete band on the agarose gel (see image below).
The monoclonal antibody must be present at a concentration of at least 0.5 g/dL in order to be accurately identified using SPEP. This corresponds to approximately 109 antibody-producing cells.  Plasma cell disorders are typically associated with the presence of an M-protein (see Table 2 below). In addition, an M component may be detected in other lymphoid malignancies like chronic lymphocytic leukemia, any B- or T- cell lymphomas, breast cancer, colon cancer, cirrhosis, sarcoidosis, and other autoimmune disorders.
Conditions associated with a monoclonal increase in the gamma region are as follows:
Monoclonal gammopathy of undetermined significance (MGUS)
Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS syndrome)
Heavy chain deposition disease
Light chain deposition disease
Infectious, Inflammatory or various reactive processes may be associated with a broad-based peak or band in the gamma region (Figure 4). This pattern suggests a polyclonal increase in immunoglobulins. Liver disease, autoimmune disease, chronic viral or bacterial infections and various malignancies may cause a polyclonal rise in the gamma fraction (see Table 2 below).
Table 2. Diseases Associated with a Polyclonal Gammopathy (Open Table in a new window)
|Liver disease||Cirrhosis, autoimmune or viral hepatitis|
|Connective tissue diseases||Rheumatoid arthritis, systemic lupus erythematosus, scleroderma, Sjogren syndrome|
Bacterial: osteomyelitis, endocarditis, osteomyelitis
Viral: HIV/AIDS, hepatitis C, Epstein-Barr virus
|Hematologic disorders/malignancies||Non-Hodgkin lymphoma, chronic lymphocytic leukemia, thalassemia, sickle cell anemia|
|Nonhematologic malignancies||Lung, ovarian, gastric malignancies, hepatocellular carcinoma|
The presence of an M-protein may be missed if the level is too small to be detected using SPEP. In cases where a high suspicion for a clonal plasma cell disorder is high, more sensitive tests such as a serum immunofixation or free light chain assay should be performed. In addition, when either alpha-1 antitrypsin deficiency or immunoglobulin deficiency, specific quantitation is indicated as SPEP is insensitive in these cases.
Collection and Panels
Container: Red-top tube, serum separator tube (SST; see the image below)
Collection method: Routine venipuncture
Blood must first be collected in a red top or serum separator tube. Serum, which is the fluid portion of blood after it has clotted, is placed on a specific medium and exposed to an electric current to separate its protein components. Two methods are usually used namely, agarose gel electrophoresis, or less commonly, capillary zone electrophoresis. [2, 4, 5, 6] In both methods, proteins are classified by their final position into five general regions:
Albumin represents the highest peak in serum protein electrophoresis, usually seen as a single, tall band. Occasionally, however two equally staining bands, referred to as bisalbuminemia or a widely staining band may be seen, which both represent normal variations.  Common conditions associated with decreased albumin include malnutrition, cirrhosis and nephrotic syndrome. Dehydration, on the other hand, causes a high albumin.
An area between albumin and the alpha-1 band is called the albumin-alpha-1 interzone. Even staining in this zone is caused by alpha-1 lipoprotein (High-density lipoprotein or HDL). An increase is usually seen in alcoholic liver disease, pregnancy and during puberty. A sharp band may also be seen in those with hepatocellular carcinoma as a result of elevated alpha-fetoprotein (AFP). 
The alpha-1 fraction includes alpha-1 antitrypsin, transcortin, and thyroid-binding globulin.  Alpha-2 fraction is comprised of ceruloplasmin, alpha-2 macroglobulin, and haptoglobin. Both alpha-1 and 2 represent the acute phase reactants; hence, malignancy, infection or any inflammatory condition can cause their elevation. A relative increase in alpha-2 fraction may be seen in nephrotic syndrome due to the relative larger size of the proteins and the inability to pass through the glomeruli. A decrease in alpha-1 component may be seen in alpha-1 antitrypsin deficiency and a decrease in alpha-2 component may be seen in hemolytic anemia due to decreased haptoglobin levels.
The beta zone consist of beta-1 and beta-2 but is often represented a graphically as a single band. Beta-1 consists mostly of transferrin, and is increased in conditions such as iron-deficiency anemia, pregnancy and estrogen therapy. B-lipoprotein and C3 complement are included in the B-2 component.  IgA, IgM, and sometimes IgG can occasionally be identified in the beta fraction as well.
Immunoglobulins mainly comprise this area including IgG, IgA, IgM, IgD and IgE. Agammaglobulinemia and hypogammaglobulinemia syndromes such as IgA deficiency are associated with a decrease in this area. Various inflammatory, autoimmune and hematologic, and non-hematologic diseases are associated with an increased gamma peak. However, a homogenous, spike-like increase in the gamma region is of special interest as it may represent an abnormal expansion of immunoglobulin-producing plasma cells.
Serum protein electrophoresis (SPEP) is an easy, inexpensive method of separating proteins based on their net charge, size, and shape. The 2 major types of protein present in the serum are albumin and the globulin proteins. Albumin is the major protein component of serum and represents the largest peak that lies closest to the positive electrode.  Globulins comprise a much smaller fraction of the total serum protein but represent the primary focus of interpretation of serum protein electrophoresis. Five globulin categories are represented: alpha-1, alpha-2, beta-1, beta-2, and gamma, with the gamma fraction being closest to the negative electrode (see the image below).
Serum protein electrophoresis is generally considered in any patient with an elevated total protein, especially those with elevated globulin level relative to albumin, or any signs and symptoms suggestive of an underlying plasma cell disorder such as multiple myeloma, Waldenstrom’s macroglobulinemia, or primary amyloidosis.  These include any of the following:
Unexplained anemia, back pain, bone pain, fatigue
Unexplained pathologic fracture or lytic lesions
Unexplained peripheral neuropathy
Hypercalcemia secondary to possible malignancy
Rouleaux formation noted on peripheral blood smear
Renal insufficiency with bland urine sediment
Bence Jones proteinuria