Serum protein electrophoresis

Serum protein electrophoresis 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 where the blood serum (the fluid portion of the blood after the blood has clot) is placed on special paper treated with agarose gel and exposed to an electric current to separate the serum protein components into five classifications by size and electrical charge, those being serum albumin, alpha-1-globulins, alpha-2-globulins, beta globulins, and gamma globulins.

Albumin
A fall of 30% is necessary before the decrease shows on electrophoresis. Usually a single band is seen. Heterozygous individuals may produce bisalbuminaemia - 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. Increased 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 is rare. This condition is called analbuminaemia. A decreased level of albumin, however, is common in many diseases and is especially important in liver disease.

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 hepatomas may result in a sharp band between the albumin and the alpha-1 zone.

Alpha-1 Zone
Orosmucoid and antitrypsin migrate together but orosmucoid stains poorly so alpha-1 antitrypsin 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 leading to emphysema caused by damaged lungs from elastase.

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 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 and therefore there is a typical increase in alpha-2 inflammation. A normal alpha-2 and a raised alpha-1 is typical in hepatic metastases 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 is raised in children and the elderly. This is seen as a sharp front to the alpha-2 band. It is of little diagnostic significance but is markedly raised in association with glomerilar protein loss - the characteristic increased alpha-2 of the nephrotic syndrome, due to its large size, cannot pass through glomeruli.

Alpha-2 globulins can be raised in cirrhosis, diabetes, and malignancy.

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.

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

Beta Zone
Transferrin comprises the beta-1 band. Increased beta-1 protein (due to the increased level of free transferrin) is typical of Iron deficiency anemia, pregnancy, and oestrogen therapy.

Beta-2 comprises C3 (Complement protein 3). It is raised in the acute phase response.

Gamma Zone
The immunoglobulins are the only proteins present in the normal gamma region. IgA has the most anodal mobility and migrates in the beta-gamma region. High levels give rise to beta/gamma fusion as in cirrhosis, respiratory infection, skin disease, or rheumatiod arthritis. IgA deficiency occurs in 1:500 of the population, as is suggested by a pallor in the gamma zone.

Commonest causes of hypergammaglobulinaemia detected by electrophoresis are severe infection, chronic liver disease, systemic lupus erythematosus, and rheumatoid arthritis.

Hypogammaglobulinaemia is easily identifiable. It is normal in infants.

Zones or faint bands in the gamma region are often seen as a result of clones of immunoglobulins responding to antigenic challenge, e.g. in chronic hepatitis and chronic viral infections.

C reactive protein can be seen as a faint band when the level is grossly abnormal, e.g. in tissue damage.

Dense narrow bands composing of immunoglobulins of a single class and containing only one type of light chain are known as paraproteins or M (monoclonal) proteins. IgA paraproteins migrate most anodally and may be confused with beta globulins. Paraproteins can arise from benign or malignant clones of B cells and their recognition and investigation is very important.

Lysozyme may be seen as a band cathodal to the slowest gamma in myelomonocytic leukaemia in which it is released from tumour cells.

Myeloma is the commonest cause of IgA and IgG paraproteinaemias but chronic lymphatic leukaemia and lymphosarcoma are not uncommon and usually give rise to IgM paraproteins. Waldenstrom's macroglobulinaemia, a malignant lymphoma, also gives rise to an IgM paraprotein.

Benign paraproteins are usually faint and do not show immunoparesis. Faint bands seen in the gamma region may be due to light chain disease, particularly if there is immune peresis.

Fibrinogen from plasma samples will be seen in the fast gamma region.