Protein Electrophoresis and Immunofixation Electrophoresis
If your doctor suspects that you have a condition that affects the antibody concentrations in the blood either by abnormal production or excessive loss
Protein electrophoresis is a method for separating the proteins found in blood (serum) or urine. During the test, an electric current is used to move the proteins across a thin layer of agarose gel. The distances that individual proteins travel depend on their size, shape, and electrical charge. These separated proteins may be detected by the use of a dye that binds to (stains) all of the proteins and reveals a characteristic pattern of bands. Each band indicates the presence of a particular protein or group of proteins, while the size of the band is a rough indication of the quantity. This pattern of bands is converted into a visual graph, showing vertical spikes or peaks where there is a lot of protein and smaller peaks or valleys where there is less. A newer method called capillary zone electrophoresis (CZE) separates proteins by passing them through a long, thin column, producing a graph that is very similar to the one made by running the protein through an agarose gel.
Specific proteins of interest can be identified by separating the proteins by gel electrophoresis and then adding an antibody. The antibody can recognise individual proteins and sticks to them making a big complex that gets caught in the gel. All the other proteins in the system are washed away and the gel stained so the proteins in question can be identified. This procedure is called immunofixation electrophoresis (IFE).
Serum proteins are separated into six major groupings by protein electrophoresis. These fractions are called albumin, alpha 1, alpha 2, beta 1, beta 2 and gamma. Albumin, which is produced in the liver, forms its own group and accounts for about 60% of the protein in the blood. 'Globulins' is a collective term used to refer to proteins other than albumin. With the exception of the immunoglobulins and some complement proteins, most of the globulins are produced in the liver. These groups are described more fully in the table below, Protein Groups.
The bands seen on protein electrophoresis form characteristic patterns. Alterations to these patterns are associated with a variety of different diseases and conditions. For example in multiple myeloma (a cancer of certain types of white blood cells called plasma cells), the uncontrolled growth and division of a malignant plasma cell leads to the production of large amounts of a single type of immunoglobulin (antibody). In contrast to other proteins in serum, which are typically of a single type, antibodies (immunoglobulins) must differ from each other to be able to recognise bacteria, viruses and other 'foreign' substances. Each time the body is exposed to a virus, for example, one plasma cell replicates and makes a group (or clone) of plasma cells to produce antibody to eliminate it. Since our total immunoglobulin represents antibody made by many clones, we refer to it as a polyclonal pattern. When there is a cancer of plasma cells, only one type of antibody is produced, termed a monoclonal pattern. This abnormal protein can be seen as a characteristic band on the electrophoresis gel.
How is the sample collected for testing?
Is any test preparation needed to ensure the quality of the sample?
No test preparation is needed.
How is it used?
Electrophoresis is used to identify the presence or absence of abnormal proteins and to identify when different groups of proteins are increased or decreased in serum or urine. It is frequently used to detect and identify monoclonal proteins (an excessive production of one specific immunoglobulin). Protein and immunofixation electrophoresis are used to help detect, diagnose, and monitor the course and treatment of conditions associated with these abnormal proteins, including multiple myeloma and a few related diseases.
Protein is usually excreted in the urine in very small amounts. When it is present in moderate to large amounts, it often indicates a problem with the kidneys. The primary reason protein and immunofixation electrophoresis are requested on urine is to look for monoclonal protein production. This protein may show up in both the serum and urine, or it may only be seen in the urine. An example of this is Bence Jones protein, which is the free light chain component of antibodies (normally, antibodies are composed of four parts, two identical heavy chains and two identical light chains. Sometimes, in multiple myeloma, only one or the other is produced, or it may be produced in excess.) The small size of Bence Jones protein allows it to pass through the kidneys and enter the urine.
Urine protein electrophoresis may also be used to help diagnose the cause and estimate the severity of protein excretion due to kidney damage or disease. This damage or disease may be due to diabetes, chronic inflammation, an autoimmune condition, or a malignancy. Electrophoresis is not usually necessary to assess the loss of small to moderate amounts of protein due to temporary conditions, such as a urinary tract infection or an acute inflammation.
When is it requested?
Protein electrophoresis may be requested when a doctor is investigating symptoms that suggest multiple myeloma, such as bone pain, anaemia, tiredness, unexplained fractures, and recurrent infections. It may also be used as a follow-up to other laboratory tests, such as an abnormal total protein and/or albumin level, elevated urine protein levels, elevated calcium levels, and low white or red blood cell counts. Immunofixation electrophoresis is usually ordered when the protein electrophoresis test shows the presence of an abnormal protein band that may be an immunoglobulin.
Electrophoresis tests are most frequently requested when a doctor suspects a disease or condition that causes a monoclonal protein to be produced. Once a disease or condition has been diagnosed, electrophoresis may be used at regular intervals to monitor the course of the disease and the effectiveness of treatment. As disease progresses, the amount of protein goes up; with treatment, it goes down. Monoclonal protein production may be due to a malignant disease, such as multiple myeloma, but it may also be due to a monoclonal gammopathy of undetermined significance (MGUS). Most patients with MGUS have no symptoms, but they must continue to be monitored regularly as some may develop multiple myeloma after a number of years.
Serum protein electrophoresis may also be used when symptoms suggest an inflammatory condition, an autoimmune disease, an acute or chronic infection, a kidney or liver disorder, or a protein-losing condition, even if the total protein and/or albumin concentrations are apparently normal. Urine protein electrophoresis may be used when there is protein detected in the urine or when the doctor suspects a monoclonal protein may be present.
What does the test result mean?
Protein and immunofixation electrophoresis tests give your doctor a rough estimate of how much of each protein is present. The value of protein electrophoresis lies in the proportions of proteins and in the patterns they create on the electrophoresis graph. The value of immunofixation electrophoresis is in the identification of the presence of a particular type of immunoglobulin.
For example, certain conditions or diseases may be associated with decreases or increases in various serum proteins, as reflected below:
- Decreased with malnutrition and malabsorption, pregnancy, kidney disease (especially nephrotic syndrome), liver disease, inflammatory conditions, and protein-losing syndromes
- Increased with dehydration
- Decreased in congenital emphysaema (a1-antitrypsin deficiency, a rare genetic disease) or severe liver disease
- Increased in acute or chronic inflammatory diseases
- Decreased with hyperthyroidism or severe liver disease, haemolysis (red blood cell breakage)
- Increased with kidney disease (nephrotic syndrome), acute or chronic inflammatory disease
- Decreased with malnutrition, cirrhosis
- Increased with hypercholesterolaemia, iron deficiency anaemia, some cases of multiple myeloma or MGUS
- Decreased variety of genetic immune disorders, and in secondary immune deficiency
- Increased Polyclonal: chronic inflammatory disease, rheumatoid arthritis, systemic lupus erythematosus, cirrhosis, chronic liver disease, acute and chronic infection, recent immunization. Monoclonal: Waldenstrom's macroglobulinaemia, multiple myeloma, monoclonal gammopathies of undetermined significance (MGUS)
Is there anything else I should know?
Immunisations within the previous six months can increase immunoglobulins as can drugs such as phenytoin (Dilantin), procainamide, oral contraceptives, methadone, and therapeutic gamma globulin.
Aspirin, bicarbonates, chlorpromazine (Thorazine), corticosteroids, and neomycin can affect protein electrophoresis results.
Why might my doctor do an electrophoresis when my total protein and albumin are normal?
They may do a serum electrophoresis because you may have an abnormality even though the total protein and albumin are normal. This is because the body tries to maintain a constant amount of protein and may increase or decrease its production of other proteins to compensate for a deficiency or overproduction of others. Your doctor may request a urine electrophoresis when there is protein in your urine, because you may lose protein in your urine even if your blood levels are fairly stable.
Is electrophoresis used for anything else?
Yes, any time separation of molecules is desired. DNA electrophoresis, for instance, is used to help study the genetic makeup of plants, animals, and humans.