Protein electrophoresis

Definition

Protein electrophoresis is a technique used to separate the different component proteins (fractions) in a mixture of proteins, such as a blood sample, on the basis of differences in how the components move through a fluid-filled matrix under the influence of an applied electric field.

Purpose

Protein electrophoresis is a screening test used to evaluate, diagnose, and monitor a variety of diseases and conditions through examination of the amounts and types of protein in a blood, urine, or cerebrospinal fluid (CSF) specimen.

Precautions

Certain other diagnostic tests or prescription medications can affect the protein electrophoresis results. The administration of a contrast dye used in some other tests may falsely elevate apparent protein levels. Drugs that can alter results include aspirin, bicarbonates, chlorpromazine (Thorazine), corticosteroids , isoniazid (INH), and neomycin (Mycifradin). The total serum protein concentration may also be affected by changes in the patient's posture or by the use of a tourniquet during the drawing of blood.

Because there is less protein in urine and CSF samples than in blood, these samples often must be concentrated before analysis. The added sample handling can lead to contamination and erroneous results. In collection of a CSF specimen, it is important that the sample not be contaminated with blood proteins that would invalidate the CSF protein measurements.

Description

Proteins—long chains of connected amino acids— are biologically important building-block chemicals that contain the elements carbon, hydrogen, nitrogen, and oxygen. Some proteins also contain sulfur, phosphorus, iron, iodine, selenium, or other trace elements. There are 22 amino acids commonly found in all proteins. The human body is capable of producing fourteen of these amino acids; the remaining eight are called essential amino acids, and must be obtained from food. Proteins are found in muscles, blood, skin, hair, nails, and the internal organs and tissues. Enzymes and antibodies are proteins, and many hormones are proteinlike. Electrophoresis is one of a variety of techniques that can be used to fractionate (separate) protein mixtures into individual component proteins.

The serum protein electrophoresis test requires a blood sample drawn by venipuncture (having blood drawn from a vein) performed in the doctor's office or on site at a medical laboratory. The urine protein electrophoresis test requires either an early morning urine sample or a 24-hour urine sample, according to the physician's request. A CSF specimen must be collected by lumbar puncture (spinal tap), generally performed by a physician as an outpatient procedure in a hospital. Because of risks associated with the lumbar-puncture procedure, the patient must sign a consent form, and should be prepared to remain for six to eight hours under observation.

Preparation

It is usually not necessary for the patient to restrict food or fluids before blood is drawn for a serum protein electrophoresis test; a four-hour fast is requested before drawing blood for lipoprotein testing. For protein electrophoresis on all types of samples, any factors that might affect test results, such as whether the patient is taking any medications, should be noted.

Aftercare

After a blood sample is drawn, a small bandage may be applied to the puncture site, and the patient may be cautioned about the possibility of fainting or of light-headedness. Following lumbar puncture for the collection of CSF, the patient must be kept lying flat in the hospital under observation for at least six to eight hours.

Risks

Risks posed by the venipuncture are minimal but may include slight bleeding from the puncture site, the development of a small bruise at the puncture site, or both. Other risks include fainting or lightheadedness after the sample is drawn. Lumbar puncture can lead to leakage of CSF from the puncture site, headache, infection, symptoms of meningitis, nausea, vomiting, or difficulty urinating. Rarely, pre-existing intracranial pressure can lead to brain herniation, resulting in brain damage or death.

Normal Results

Blood proteins

Serum protein electrophoresis is used to determine the total serum protein concentration, which is an indication of the patient's hydration state: dehydration leads to high total serum protein concentration. Further, the levels of different blood proteins rise or fall in response to such disorders as cancer and associated protein-wasting syndromes, immune-system disorders, liver dysfunction, impaired nutrition, and chronic fluid-retaining conditions. The different types of blood proteins are separated into fractions of five distinct classes: albumin, alpha ₁-globulins, alpha ₂-globulins, beta-globulins, and gamma-globulins (immunoglobulins). In addition to standard protein electrophoresis, immunoelectrophoresis may be used to assess the blood levels of specific immunoglobulins. Immunoelectrophoresis is usually ordered when the serum protein electrophoresis test shows an unusually high amount of protein in the gamma-globulin fraction.

ALBUMIN.

Albumin, which is produced in the liver, is the most abundant blood protein. It makes a major contribution to the regulation of water movement between the tissues and the bloodstream. Albumin binds calcium, thyroid hormones, fatty acids, and many drugs, keeping them in the blood circulation and preventing them from being filtered out by the kidneys. Albumin levels can play a role in the effectiveness and toxicity of therapeutic drugs and in drug interactions.

GLOBULINS.

Serum globulins are separated in protein electrophoresis as four main fractions: alpha ₁-, alpha ₂-, beta-, and gamma-globulins.

• The major alpha ₁-globulin is alpha ₁-antitrypsin, produced by the lungs and liver. Alpha ₁-antitrypsin deficiency is a marker of an inherited disorder characterized by an increased risk of emphysema.
• Alpha ₂-globulins include serum haptoglobin, alpha ₂-macroglobulin, and ceruloplasmin. Haptoglobin binds to hemoglobin, released from damaged red blood cells during hemolysis, to prevent its excretion by the kidneys. Alpha ₂-macroglobulin accounts for about one third of the alpha ₂-globulin fraction. Ceruloplasmin is involved in the storage and transport of copper and iron in the body.
• Beta-globulins include transferrin, low-density lipoproteins (LDL), and complement components. Transferrin transports dietary iron to the liver, spleen, and bone marrow. Low-density lipoprotein is the major carrier of cholesterol in the blood. Complement is a system of blood proteins involved in inflammatory response. (See Key Terms)
• The gamma-globulin fraction contains the immunoglobulins, a family of proteins that function as antibodies. Antibodies, in response to infection, allergic reactions, and organ transplants, recognize and bind foreign bodies, or antigens, to facilitate their destruction by the immune system. The immune response is regulated by a large number of antigen-specific gamma-globulins that fall into five main classes called IgG, IgA, IgM, IgB, and IgE. When the serum protein electrophoresis test demonstrates a significant deviation from the normal gamma-globulin levels, a supplemental test, immunoelectrophoresis, should be ordered to identify the specific globulin (s) involved.

The following serum protein electrophoresis reference values are representative; some variation among laboratories and specific methods is to be expected. (1 gm = approximately 0.02 pt and 1 dl = approximately 0.33 fluid oz.)

• Total protein: 6.4-8.3 g/dL
• Albumin: 3.5-5.0 g/dL
• Alpha ₁-globulin: 0.1-0.3 g/dL
• Alpha ₂ globulin: 0.6-1.0 g/dL
• Beta-globulin: 0.7-1.2 g/dL
• Gamma-globulin: 0.7-1.6 g/dL

Urinary proteins

Protein electrophoresis is performed on urine samples to classify disorders that cause protein loss via the kidneys. In urine, normally no globulins and less than 0.050 g/dL albumin are present.

Cerebrospinal fluid (CSF) proteins

In CSF, the total protein concentration is normally 0.015-0.045 g/dL, with gamma-globulin accounting for 3% to 12%. The main use of CSF protein electrophoresis testing is in the diagnosis of central nervous tumors and multiple sclerosis.

Abnormal results

Deviations in serum protein levels from reference levels are considered in conjunction with symptoms and results from other diagnostic procedures.

Albumin levels are increased in dehydration and decreased in malnutrition, pregnancy, liver disease, inflammatory diseases, and protein-losing states such as malabsorption syndrome and certain kidney disorders. Low serum albumin levels can indicate disease and can influence analysis of thyroid hormones and calcium.

Alpha ₁-globulins are increased in inflammatory diseases and decreased or absent in juvenile pulmonary emphysema, a hereditary disease.

Alpha ₂-globulins are increased in acute and chronic inflammation and nephrotic syndrome. Decreased values may indicate hemolysis (the release of hemoglobin from red blood cells). Low haptoglobin can indicate tumor metastasis , severe sepsis, or chronic liver disease. The concentration of macroglobulin is increased during nephrosis. Ceruloplasmin concentration is increased during pregnancy and decreased in Wilson's disease, a rare inherited condition that leads to accumulation of copper in the liver.

Beta-globulin levels are increased in multiple myeloma and also in conditions of high cholesterol (hypercholesterolemia), such as in atherosclerosis, and in iron deficiency anemia . Levels are decreased in coagulation disorders.

Gamma-globulin levels are increased in multiple myeloma. The levels are increased as well in chronic inflammatory disease and autoimmune conditions such as rheumatoid arthritis and systemic lupus erythematosus, cirrhosis, and acute and chronic infection. The gamma-globulins are decreased in leukemia, in a variety of genetic immune disorders, and in secondary immune deficiency related to steroid use or to severe infection. Immunoglobulin deficiency due to inherited disorders can range from partial or complete loss of a single immunoglobulin class to complete absence of all immunoglobulins.

Finding an individual (oligoclonal) band in the gamma fraction of the electrophoresis result indicates the presence of a paraprotein. Type IgG or IgA paraproteins associated with multiple myeloma may be found by serum protein electrophoresis testing; however, the tumor may also produce only Ig light chains that are removed from the blood by the kidneys. This Ig light chain (also known as the Bence-Jones protein) is detected by urine protein electrophoresis and is found nearly exclusively in patients with multiple myeloma.

In urine samples, abnormal results other than the presence of the Bence-Jones protein indicate disruption of kidney function or acute inflammation. Hemoglobin and myoglobin are found in the urine of patients with infection or hemolysis.

An increase in total protein concentration in the CSF is often found with central nervous system (CNS) tumors and in meningitis.

Resources

BOOKS

Marshall, W.J. Clinical Chemistry, 4th ed. Edinburgh, London, New York, Philadelphia, St. Louis, and Toronto: Mosby, 2000.

Handbook of Capillary Electrophoresis Applications. Shintani, H., and J. Polonsky, eds. London: Blackie Academic and Professional, 1998.

OTHER

Health Wide Web site. 2000 Healthwide.com, Inc. <http://www.healthwide.com/ency>.

Patricia L. Bounds, Ph.D.

KEY TERMS

Acute-phase proteins

—Proteins produced during the acute-phase response, a set of physiological changes that occur in response to biologic stress such as trauma or sepsis.

Albumin

—A blood protein produced in the liver that helps to regulate water distribution in the body.

Antibodies

—Immunoglobulin protein molecules produced by B-cells during the immune response. Each antibody recognizes an individual antigen to trigger immune defenses.

Antigen

—Foreign body that triggers immune response.

Bence-Jones protein

—The Ig light chain, part of an immunoglobulin, that is detected by urine protein electrophoresis in the case of multiple myeloma.

Complement

—A group of complex proteins of the beta-globulin type in the blood that bind to antibodies during anaphylaxis. In the complement cascade, each complement interacts with another in a pattern that causes fluid build-up in cells, leading to lysis (cell destruction).

Electrophoresis

—A technique used to separate the proteins in a biological sample on the basis of differences in how the components move through a fluid-filled matrix under the influence of an applied electric field.

Globulins

—A group of proteins in blood plasma whose levels can be measured by electrophoresis in order to diagnose or monitor a variety of serious illnesses.

Hemolysis

—Also called hematolysis, the breakage of red blood cells and concomitant liberation of hemoglobin.

Lumbar puncture

—Also called spinal tap, a procedure for the withdrawal of spinal fluid from the lumbar region of the spinal cord for diagnosis, or for injection of a dye for imaging, or for administering medication or an anesthetic.

Paraprotein

—A paraprotein is an immunoglobulin produced by a clone of identical B-cells.

Protein

—Proteins, such as enzymes and antibodies, are biologically important molecules made of long chains of connected amino acids that contain the elements carbon, hydrogen, nitrogen, and oxygen. Certain proteins may also contain sulfur, phosphorus, iron, iodine, selenium, or other trace elements.

Protein Electrophoresis

Definition

Electrophoresis is a technique used to separate different elements (fractions) of a blood sample into individual components. Serum protein electrophoresis (SPEP) is a screening test that measures the major blood proteins by separating them into five distinct fractions: albumin, alpha₁, alpha₂, beta, and gamma proteins. Protein electrophoresis can also be performed on urine.

Purpose

Protein electrophoresis is used to evaluate, diagnose, and monitor a variety of diseases and conditions. It can be used for these purposes because the levels of different blood proteins rise or fall in response to such disorders as cancer, intestinal or kidney protein-wasting syndromes, disorders of the immune system, liver dysfunction, impaired nutrition, and chronic fluid-retaining conditions.

Precautions

Certain other diagnostic tests or prescription medications can affect the results of SPEP tests. The administration of a contrast dye used in some other tests may falsely elevate protein levels. Drugs that can alter results include aspirin, bicarbonates, chlorpromazine (Thorazine), corticosteroids, isoniazid (INH), and neomycin (Mycifradin).

Description

Proteins are major components of muscle, enzymes, hormones, hemoglobin, and other body tissues. Proteins are composed of elements that can be separated from one another by several different techniques: chemical methods, ultracentrifuge, or electrophoresis. There are two major types of electrophoresis: protein electrophoresis and immunoelectrophoresis. Immunoelectrophoresis is used to assess the blood levels of specific types of proteins called immunoglobulins. An immunoelectrophoresis test is usually ordered if a SPEP test has a "spike," or rise, at the immunoglobulin level. Protein electrophoresis is used to determine the total amount of protein in the blood, and to establish the levels of other types of proteins called albumin, alpha₁ globulin, alpha₂ globulin, and beta-globulin.

Blood proteins

ALBUMIN. Albumin is a protein that is made in the liver. It helps to retain elements like calcium, some hormones, and certain drugs in the circulation by binding to them to prevent their being filtered out by the kidneys. Albumin also acts to regulate the movement of water between the tissues and the bloodstream by attracting water to areas with higher concentrations of salts or proteins.

GLOBULINS. Globulins are another type of protein, larger in size than albumin. They are divided into three main groups: alpha, beta, and gamma.

• Alphaglobulins. These proteins include alpha₁ and alpha₂ globulins. Alpha₁ globulin is predominantly alpha₁ antitrypsin, an enzyme produced by the lungs and liver. Alpha₂ globulin, which includes serum haptoglobin, is a protein that binds hemoglobin to prevent its excretion by the kidneys. Various other alphaglobulins are produced as a result of inflammation, tissue damage, autoimmune disorders, or certain cancers.
• Betaglobulins. These include low-density substances involved in fat transport (lipoproteins), iron transport (transferrin), and blood clotting (plasminogen and complement).
• Gammaglobulins. All of the gammaglobulins are antibodies—proteins produced by the immune system in response to infection, allergic reactions, and organ transplants. If serum protein electrophoresis has demonstrated a significant rise at the gammaglobulin level, immunoelectrophoresis is done to identify the specific globulin that is involved.

Electrophoretic measurement of proteins

All proteins have an electrical charge. The SPEP test is designed to make use of this characteristic. There is some difference in method, but basically the sample is placed in or on a special medium (e.g., a gel), and an electric current is applied to the gel. The protein particles move through the gel according to the strength of their electrical charges, forming bands or zones. An instrument called a densitometer measures these bands, which can be identified and associated with specific diseases. For example, a decrease in albumin with a rise in the alpha₂ globulin usually indicates an acute reaction of the type that occurs in infections, burns, stress, or heart attack. On the other hand, a slight decrease in albumin, with a slight increase in gammaglobulin, and a normal alpha₂ globulin is more indicative of a chronic inflammatory condition, as might be seen in cirrhosis of the liver.

Protein electrophoresis is performed on urine samples to classify kidney disorders that cause protein loss. Here also certain band patterns are specific for disease. For example, the identification of a specific protein called the Bence Jones protein (by performing the Bence Jones protein test ) during the procedure suggests multiple myeloma.

Preparation

The serum protein electrophoresis test requires a blood sample. It is not necessary for the patient to restrict food or fluids before the test. The urine protein electrophoresis test requires either an early morning urine sample or a 24-hour urine sample according to the physician's request. The doctor should check to see if the patient is taking any medications that may affect test results.

Risks

Risks posed by the blood test are minimal but may include slight bleeding from the puncture site, fainting or lightheadedness after the blood is drawn, or the development of a small bruise at the puncture site.

Normal results

The following values are representative, although there is some variation among laboratories and specific methods. These values are based on the agarose system.

• total protein: 5.9-8.0 g/dL
• albumin: 4.0-5.5 g/dL
• alpha₁ globulin: 0.15-0.25 g/dL
• alpha₂ globulin: 0.43-0.75 g/dL
• beta-globulin: 0.5-1.0 g/dL
• gammaglobulin: 0.6-1.3 g/dL

Abnormal results

Albumin levels are increased in dehydration. They are decreased in malnutrition, pregnancy, liver disease, inflammatory diseases, and such protein-losing syndromes as malabsorption syndrome and certain kidney disorders.

Alpha₁ globulins are increased in inflammatory diseases. They are decreased or absent in juvenile pulmonary emphysema, which is a genetic disease.

Alpha₂ globulins are increased in a kidney disorder called nephrotic syndrome. They are decreased in patients with an overactive thyroid gland (hyperthyroidism ) or severe liver dysfunction.

Betaglobulin levels are increased in conditions of high cholesterol levels (hypercholesterolemia ) and iron deficiency anemia. They are decreased in malnutrition.

Gammaglobulin levels are increased in chronic inflammatory disease (for example, rheumatoid arthritis, systemic lupus erythematosus); cirrhosis; acute and chronic infection; and a cancerous disease characterized by uncontrolled multiplication of plasma cells in the bone marrow (multiple myeloma). Gammaglobulins are decreased in a variety of genetic immune disorders, and in secondary immune deficiency related to steroid use, leukemia, or severe infection.

Resources

BOOKS

Pagana, Kathleen Deska. Mosby's Manual of Diagnosticand Laboratory Tests. St. Louis: Mosby, Inc., 1998.

KEY TERMS

Albumin— A blood protein that is made in the liver and helps to regulate water movement in the body.

Electrophoresis— A technique for separating various blood fractions by running an electric current through a gel containing a blood sample.

Globulins— A group of proteins in blood plasma whose levels can be measured by electrophoresis in order to diagnose or monitor a variety of serious illnesses.

Haptoglobin— A protein in blood plasma that binds hemoglobin.

Immunoglobulins— Any of several types of globulin proteins that function as antibodies.