Protein Electrophoresis Test

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Protein Electrophoresis Test

Definition

Electrophoresis is a technique used to separate the different components (fractions) of a mixture, such as proteins in a biological sample. Separation is based on differences in the charge (and sometimes size) of the molecules, which determines their rate of movement in an electric field. Serum protein electrophoresis is a screening test that measures the major blood proteins. Protein electrophoresis testing can also be performed on urine and cerebrospinal fluid (CSF) samples.

Purpose

Protein electrophoresis testing is 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 CSF specimen.

Electrophoresis results	Disease
Source: Pagana, K.D. and T.J. Pagana. Mosby's Diagnostic and Laboratory Test Reference. 3rd ed. St. Louis: Mosby, 1997.
Total protein: 6.4-8.3 g/dl (64.0-83.0 g/L)	Normal results
Albumin: 3.5-5.0 g/dl (35-50 g/L)
Alpha1 globulin: 0.1-0.3 g/dl (1-3 g/L)
Alpha₂ globulin: 0.6-1.0 g/dl (6-10 g/L)
Beta globulin: 0.7-1.1 g/dl (7-11 g/L)
Decreased albumin Increased alpha₂ globulin	Acute infections, tissue necrosis, burns, surgery, stress, myocardial infarction
Slightly decreased albumin Slightly increased gamma gobulin Normal alpha₂ globulin	Chronic infection, granulomatus diseases, cirrhosis, rheumatoid-collegen diseases
Greatly decreased albumin Greatly increased alpha₂ globulin Normal increase in beta globulin	Nephrotic syndrome
Decreased albumin Increased gamma globulin Incorporation of beta and gamma peaks	Far-advanced cirrhosis
Greatly increased gamma globulin	Cirrhosis, chronic infection, globulin with a broad peaksarcoidosis, tuberculosis, endocarditis, rheumatoid-collagen disease
Decreased gamma globulin with normal other globulin levels	Light-chain multiple myeloma
Thin spikes in gamma globulin	Myeloma, macroglobulinemia, gammopathies

Precautions

Certain other diagnostic tests or prescription medications can affect the results of protein electrophoresis tests. 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 venipuncture.

Protein is less concentrated in urine and CSF than in blood. Urinary and CSF proteins must be concentrated before analysis, and the added sample handling can lead to contamination and erroneous results. In the 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 are biologically important organic molecules—polymers of 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. There are twenty-two amino acids commonly found in all proteins. The human body is capable of producing fourteen of these amino acids; the remaining eight so-called essential amino acids must be obtained from food. Proteins are found in muscles, blood, skin, hair, nails, and the internal organs and tissues. Enzymes, hemoglobin, and antibodies are proteins, as are many hormones.

Protein mixtures can be fractionated into individual component proteins by a variety of techniques, including precipitation, chromatography, ultracentrifugation, or electrophoresis.

A serum protein electrophoresis test is used to determine the percentage of each protein in the blood by separating them into five distinct classes: albumin, alpha₁-globulin, alpha²-globulin, beta-globulin, and gamma-globulins (immunoglobulins). High-resolution protein electrophoresis uses a higher current to separate the major proteins comprising the alpha₁-globulin, alpha₂-globulin, and beta-globulin fractions. This procedure produces nine or more bands, including alpha₁ antitrypsin, alpha₂ macroglobulin, haptoglobulin, transferrin, and complement proteins.

In addition to standard protein electrophoresis, the immunofixation electrophoresis test (IFE) may be used to assess the blood levels of specific immunoglobulins. An IFE test is usually ordered if a serum protein electrophoresis test shows an unusually high amount of protein in the gamma-globulin fraction. The IFE tests determine whether the increase in the gamma-globulin fraction is caused by excess immunoglobulins (antibodies) and whether it is polyclonal or monoclonal in nature. Polyclonal increases are caused by infections, allergies, and inflammatory diseases, while monoclonal increases are caused by malignant or benign proliferations of the antibody-producing cells (plasma cells).

Serum proteins

The total serum protein concentration may be used to assess a 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, intestinal or kidney protein-wasting syndromes, disorders of the immune system, liver dysfunction, impaired nutrition, and chronic fluid-retaining conditions.

ALBUMIN. Albumin, which is produced in the liver, is the most abundant blood protein. It makes a major contribution to the osmotic pressure that regulates the movement of water between the tissues and the bloodstream. Albumin binds calcium, thyroid hormones, fatty acids, and many drugs, maintaining them in the circulation and preventing filtration in the kidneys. Low serum albumin levels can be indicative of pathology, and can increase free therapeutic drug levels and decrease total calcium levels. Albumin levels can play a role in the efficacy and toxicity of therapeutic drugs, and in drug interactions.

GLOBULINS. Serum globulins are present in protein electropherograms as four main fractions: alpha₁-, alpha₂-, beta-, and gamma-globulins.

The major alpha₁-globulin is the "acute-phase" protein alpha1-antitrypsin, a protease inhibitor produced by the lungs and liver. Alpha₁-antitrypsin deficiency is a marker of an inherited disorder characterized by an increased risk of emphysema.
Alpha₂-globulins include serum haptoglobin, alpha₂-macroglobulin, and ceruloplasmin. Haptoglobin is an acute-phase protein that binds free hemoglobin released from red blood cells during hemolysis. Haptoglobin binding prevents excretion of free hemoglobin by the kidneys. In addition to hemolysis, low haptoglobin levels can indicate chronic liver disease, tumor metastasis, or severe sepsis. Alpha₂-macroglobulin, a broad-spectrum protease inhibitor, accounts for about one-third of the alpha₂-globulin fraction: its concentration is increased during nephrosis. Ceruloplasmin is an acute-phase protein involved in the storage and transport of copper and iron: its concentration is increased during pregnancy and decreased in Wilson's disease.
Beta-globulins include transferrin, low-density lipoproteins (LDL), and complement components. Transferrin transports dietary iron to the liver, spleen, and bone marrow. LDL (also referred to as beta lipoprotein) is the major carrier of cholesterol in the blood: high levels are associated with atherosclerosis. Complement is a system of blood proteins required for antibody-mediated cell lysis and involved in the inflammatory response.
The gamma-globulin fraction contains the immunoglobulins, a family of proteins that function as antibodies. Antibodies, produced in response to infection, allergic reactions, and organ transplants, recognize and bind antigens to facilitate 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, IgO, and IgE. Immunoglobulin deficiency due to inherited disorders can range from partial or complete loss of a single immunoglobulin class to complete absence of all immunoglobulins. An abnormally high level of immunoglobulins is generally found in acute and chronic infections, and is an indicator of autoimmune disease. When the serum protein electrophoresis test demonstrates a significant deviation from the normal gamma-globulin levels, an IFE test should be ordered to determine the polyclonal or monoclonal nature of the specific globulin(s) involved.

Quantification of each immunoglobin class and each of the proteins mentioned above may be performed by a procedure called immunonephelometry. This technique uses an antibody specific for the protein to be measured. When the antibody binds to the protein, an immune complex is formed that increases the amount of light scattered by the sample.

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

Urinary proteins

Protein electrophoresis is performed on urine samples to classify disorders that cause protein loss via the kidneys. Hemoglobin and myoglobin are found in the urine of trauma and burn victims, and in patients with infection or hemolysis. Protein electrophoresis of urine is most often performed in order to detect the presence of light chain fragments of immunoglobulins. These protein fragments are sufficiently small to filter through the kidneys and are excreted in the urine. They are called Bence-Jones proteins, and are found in patients who have multiple myeloma, a malignant proliferation of antibody-producing cells. Bence-Jones proteins may also be found in other variants of multiple myeloma, such as light chain disease, and in patients with systemic autoimmune diseases that result from degradation of immune complexes.

Cerebrospinal fluid (CSF) proteins

An increase in total protein concentration in the CSF is often found in bacterial and fungal meningitis and with central nervous system (CNS) tumors. The main use of CSF protein electrophoresis testing is in the diagnosis of multiple sclerosis.

The protein electrophoresis test method

In a clinical protein electrophoresis test, proteins are separated on the basis of how fast they move on a medium in an electrical field. In a standard electrophoresis test, a small amount of sample is applied to a cellulose acetate strip or gel made of agarose or polyacrylamide. The strip or gel is positioned between the apparatus electrodes, and a voltage is applied across it. Under the conditions of the test, the proteins in the sample are negatively charged and migrate toward the positive electrode at different rates. The migration rate is mainly dependent on the charge of the protein molecules; however, on polyacrylamide gel it is also dependent on size. When electrophoresis is complete, the gel is treated with a dye to stain the proteins, and the intensity of stain in the bands is measured and recorded by a densitometer.

In a capillary electrophoresis test, samples are automatically transferred from the collection tubes to the head of a fluid-filled glass capillary tube: the electrophoretic separation occurs during the transport of the proteins through the capillary, and the individual proteins are detected and quantified on-line by absorption of ultraviolet light. Both gel and capillary electrophoresis procedures produce a recording of each protein fraction as a peak. The area under the peak is used to calculate the percentage of the fraction. This is multiplied by the total protein concentration (determined by chemical analysis) to give the concentration of each fraction. The levels of proteins thus determined are compared with reference levels to ascertain whether a disease state is present.

Standard electrophoresis systems

High-throughput and semi-automated protein electrophoresis testing is available in most laboratories through the use of integrated systems of gels, reagent kits, and densitometers.

Capillary electrophoresis systems

In 1998, the introduction of fully automated clinical capillary electrophoresis instruments was introduced. The advantages of the capillary electrophoresis methodology include direct sampling of a minimal volume from the primary collection tube, automated detection of proteins without staining, rapid sample throughput, and improved accuracy and reproducibility. Agarose and capillary systems are also used to separate DNA, an increasingly important technology.

Preparation

The sample for the serum protein electrophoresis test is obtained by venipuncture. No anticoagulant should be used. It is usually not necessary for the patient to restrict food or fluids before the test; a 12-hour fast is requested before drawing blood for lipoprotein testing. The urine protein electrophoresis test requires either an early morning urine sample or a 24-hour urine sample according to the physician's request. CSF is collected by lumbar puncture performed in a hospital setting; because of risks associated with the procedure, the patient must sign a consent form. Any factors that might affect test results, such as whether the patient is taking any medications, should be noted.

Complications

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

Lumbar puncture can lead to leakage of cerebrospinal fluid 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 and/or death. The patient must be kept lying flat in the hospital under observation for at least six to eight hours after the procedure.

Results

The following serum protein electrophoresis reference values are representative; some variation among laboratories and specific methods is to be expected. The values were obtained by standard electrophoresis on agarose gels.

total protein: 6.4 to 8.3 g/dL (about 0.5 g/dL lower in nonambulatory patients)
albumin: 3.5 to 5.0 g/dL
alpha₁-globulin: 0.1 to 0.3 g/dL
alpha₂-globulin: 0.6 to 1.0 g/dL
beta-globulin: 0.7 to 1.2 g/dL
gamma-globulin: 0.7 to 1.6 g/dL

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.

Alpha₁-globulins are increased in inflammatory diseases and decreased or absent in juvenile pulmonary emphysema, a hereditary disease.

Alpha₂-globulins are increased in acute and chronic inflammation and nephrotic syndrome; decreased values may indicate hemolysis.

Beta-globulin levels are increased in conditions of high cholesterol (hypercholesterolemia), in multiple myeloma, and in iron deficiency anemia; and decreased in disorders associated with complement depletion.

Gamma-globulin levels are increased in chronic inflammatory disease and autoimmune conditions such as rheumatoid arthritis and systemic lupus erythematosus, cirrhosis, in acute and chronic infection, and in multiple myeloma. The gamma-globulins are decreased in a variety of genetic immune disorders, in secondary immune deficiency related to steroid use, leukemia, or severe infection.

Detection of a discrete (monoclonal) band in the gamma region of the electropherogram 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 produce only Ig light chains that are removed from the blood by the kidneys. The Ig light chain (Bence-Jones protein) can be detected by urine protein electrophoresis.

In CSF, the total protein concentration is normally 0.015 to 0.045 g/dL, with gamma-globulin accounting for 3-12%. Increased gamma-globulin indicates multiple sclerosis, bacterial or fungal meningitis, neurosyphilis, or Guillain-Barrésyndrome. Detection of monoclonal bands in CSF plays an important role in the diagnosis of multiple sclerosis.

In urine, normally no globulins and less than 0.050 g/dL albumin are present. Abnormal results indicate disruption of kidney function or acute inflammation. The presence of the Bence-Jones protein indicates multiple myoloma.

KEY TERMS

Acute-phase proteins— Proteins produced during the acute-phase response, a set of physiological changes that occur in response to trauma conditions.

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 and plasma cells during the immune response. Each antibody-binding site can bind part of an 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 component interacts with another in a cascade that causes cell membrane damage, leading to lysis.

Electropherogram— The pattern of stained proteins on an electrophoresis gel, or a graph showing peaks of proteins separated by electrophoresis.

Electrophoresis— A technique used to separate the proteins in a biological sample on the basis of charge and size as they migrate under the influence of an applied electric field.

Globulins— Plasma or serum proteins other than albumin, so named because of their globular shape. Four globulin fractions can be measured by electrophoresis in order to diagnose or monitor a variety of serious illnesses.

Paraprotein— An immunoglobulin produced by a clone of identical B-cells. Also called monoclonal protein.

Protein— Proteins, such as enzymes and antibodies, are biologically important polymers of 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.

Health care team roles

Nurses are involved in the collection of blood samples by venipuncture, in patient support during and after lumbar puncture, and in instructing patients in the procedure for midstream collection of urine samples. A lumbar puncture is generally performed by a physician. Medical laboratory workers are responsible for preparation of samples for electrophoresis testing.

Training

The preparation of urine and CSF samples for electrophoresis testing often involves concentration and, in the case of urine samples and some CSF samples, centrifugation. Electrophoresis is classified as a high-complexity test and is performed by laboratory personnel with special training, most often by a clinical laboratory scientist (CLS) or medical technologist (MT).