Serum protein electrophoresis

Schematic representation of a protein electrophoresis gel

Normal serum protein electrophoresis diagram with legend of different zones.

Serum protein electrophoresis (SPEP or SPE) is a laboratory test that examines specific proteins in the blood called globulins. Blood must first be collected, usually into an airtight vial or syringe. Electrophoresis is a laboratory technique in which the blood serum (the fluid portion of the blood after the blood has clotted) is placed into a gel, or into liquid in a capillary tube, and exposed to an electric current to separate the serum protein components into five major fractions by size and electrical charge: serum albumin, alpha-1 globulins, alpha-2 globulins, beta globulins, and gamma globulins.

Albumin

Albumin is the major fraction in a normal SPEP. A fall of 30% is necessary before the decrease shows on electrophoresis. Usually a single band is seen. Heterozygous individuals may produce bisalbuminemia - two equally staining bands, the product of two genes. Some variants give rise to a wide band or two bands of unequal intensity but none of these variants is associated with disease.^[1] Increased anodic mobility results from the binding of bilirubin, nonesterified fatty acids, penicillin and acetylsalicylic acid, and occasionally from tryptic digestion in acute pancreatitis.

Absence of albumin, known as analbuminaemia, is rare. A decreased level of albumin, however, is common in many diseases, including liver disease, malnutrition, malabsorption, protein-losing nephropathy and enteropathy.^[2]

Albumin - Alpha-1 Interzone

Even staining in this zone is due to alpha-1 lipoprotein (High density lipoprotein - HDL). Decrease occurs in severe inflammation, acute hepatitis, and cirrhosis. Also, nephrotic syndrome can lead to decrease in albumin level; due to its loss in the urine through a damaged leaky glomerulus. An increase appears in severe alcoholics and in women during pregnancy and in puberty.

The high levels of AFP that may occur in hepatocellular carcinoma may result in a sharp band between the albumin and the alpha-1 zone.

Alpha-1 Zone

Orosomucoid and antitrypsin migrate together but orosmucoid stains poorly so alpha 1 antitrypsin (AAT) constitutes most of the alpha-1 band. Alpha-1 antitrypsin has an SG group and thiol compounds may be bound to the protein altering their mobility. A decreased band is seen in the deficiency state. It is decreased in the nephrotic syndrome and absence could indicate possible alpha 1-antitrypsin deficiency. This eventually leads to emphysema from unregulated neutrophil elastase activity in the lung tissue. The alpha-1 fraction does not disappear in alpha 1-antitrypsin deficiency, however, because other proteins, including alpha-lipoprotein and orosomucoid, also migrate there. As a positive acute phase reactant, AAT is increased in acute inflammation.

Bence Jones protein may bind to and retard the alpha-1 band.

Alpha-1 - Alpha-2 Interzone

Two faint bands may be seen representing alpha-1 antichymotrypsin and vitamin D binding protein. These bands fuse and intensify in early inflammation due to an increase in alpha-1 antichymotrypsin, an acute phase protein.

Alpha-2 Zone

This zone consists principally of alpha-2 macroglobulin (AMG or A2M) and haptoglobin. There are typically low levels in haemolytic anaemia (haptoglobin is a suicide molecule which binds with free haemoglobin released from red blood cells and these complexes are rapidly removed by phagocytes). Haptoglobin is raised as part of the acute phase response, resulting in a typical elevation in the alpha-2 zone during inflammation. A normal alpha-2 and an elevated alpha-1 zone is a typical pattern in hepatic metastasis and cirrhosis.

Haptoglobin/haemaglobin complexes migrate more cathodally than haptoglobin as seen in the alpha-2 - beta interzone. This is typically seen as a broadening of the alpha-2 zone.

Alpha-2 macroglobulin may be elevated in children and the elderly. This is seen as a sharp front to the alpha-2 band. AMG is markedly raised (10-fold increase or greater) in association with glomerular protein loss, as in nephrotic syndrome. Due to its large size, AMG cannot pass through glomeruli, while other lower-molecular weight proteins are lost. Enhanced synthesis of AMG accounts for its absolute increase in nephrotic syndrome.

AMG is mildly elevated early in the course of diabetic nephropathy.

Alpha-2 - Beta Interzone

Cold insoluble globulin forms a band here which is not seen in plasma because it is precipitated by heparin. There are low levels in inflammation and high levels in pregnancy.

Beta lipoprotein forms an irregular crenated band in this zone. High levels are seen in type II hypercholesterolaemia, hypertriglyceridemia, and in the nephrotic syndrome.

Beta Zone

Transferrin and beta-lipoprotein (LDL) comprises the beta-1. Increased beta-1 protein due to the increased level of free transferrin is typical of iron deficiency anemia, pregnancy, and oestrogen therapy. Increased beta-1 protein due to LDL elevation occurs in hypercholesterolemia. Decreased beta-1 protein occurs in acute or chronic inflammation.

Beta-2 comprises C3 (Complement protein 3). It is raised in the acute phase response. Depression of C3 occurs in autoimmune disorders as the complement system is activated and the C3 becomes bound to immune complexes and removed from serum. Fibrinogen, a beta-2 protein, is found in normal plasma but absent in normal serum. Occasionally, blood drawn from heparinized patients does not fully clot, resulting in a visible fibrinogen band between the beta and gamma globulins.

Beta-Gamma Interzone

C-Reactive Protein is found in between the beta and gamma zones producing beta/gamma fusion. IgA has the most anodal mobility and migrates in the region between the beta and gamma zones also causing a beta/gamma fusion in patients with cirrhosis, respiratory infection, skin disease, or rheumatoid arthritis (increased IgA).

Gamma Zone

The immunoglobulins (IgA, IgM, IgG, IgE and IgD) are the only proteins present in the normal gamma region, but note that immunoglobulins may be found in the alpha and beta zones. If the gamma zone shows an increase (or spike), the first step in interpretation is to establish if the region is narrow or wide. If it is elevated in a single narrow "spike-like" manner it could indicate monoclonal production of a single immunoglobulin (monoclonal gammopathy), while a broad "swell-like" manner (wide) indicates polyclonal immunoglobulin production.^[3] Immunofixation (gel electrophoresis) or immunosubtraction (capillary electrophoresis) are performed to detect and confirm monoclonal immunoglobulins.

• Monoclonal Gammopathy - A narrow spike indicates a monoclonal gammopathy, also known as an "M-spike". Typically, a monoclonal gammopathy is malignant or clonal in origin, Myeloma being the most common cause of IgA and IgG spikes. chronic lymphatic leukaemia and lymphosarcoma are not uncommon and usually give rise to IgM paraproteins. Note that up to 8% of healthy geriatric patients may have a monoclonal spike.^[4] Waldenstrom's macroglobulinaemia (IgM), monoclonal gammopathy of undetermined significance (MGUS), amyloidosis, plasma cell leukemia and solitary plasmacytomas also produce an M-spike.
• Polyclonal Gammopathy - A "swell-like" elevation in the gamma zone indicates a polyclonal gammopathy, which typically indicates a non-neoplastic condition (although is not exclusive to non-neoplastic conditions). The most common causes of polyclonal hypergammaglobulinaemia detected by electrophoresis are severe infection, chronic liver disease, rheumatoid arthritis, systemic lupus erythematosus and other connective tissue diseases.
• Hypogammaglobulinaemia - This is easily identifiable as a "slump" or decrease in the gamma zone. It is normal in infants. It is found in patients with X-linked agammaglobulinemia. IgA deficiency occurs in 1:500 of the population, as is suggested by a pallor in the gamma zone.

Lysozyme may be seen as a band cathodal to the slowest gamma in myelomonocytic leukaemia in which it is released from tumour cells. Fibrinogen from plasma samples will be seen in the fast gamma region.

References

External links

• Proetin electrophoresis at Lab Tests Online