PROTEIN ELECTROPHORESIS
INTRODUCTION
Serum protein electrophoresis is a laboratory examination that commonly is used to identify patients with multiple myeloma and other disorders of serum protein. Many subspecialists include serum protein electrophoresis screening in the initial evaluation for numerous clinical conditions. Sometimes, however, the results of this examination can be confusing or difficult to interpret (Jenkins, 2019). This seminar paper discussed protein electrophoresis in details which covers the definition, Acetate or gel electrophoresis, Capillary electrophoresis, Components of Serum Protein Electrophoresis, Indications, Interpretation of Results, Monoclonal Versus Polyclonal Gammopathies and Evaluation of an Abnormal Serum Protein Electrophoresis
THE CONCEPT OF PROTEIN ELECTROPHORESIS
Serum protein electrophoresis (SPEP or SPE) is a laboratory test that examines specific proteins in the blood called globulins(Jenkins, 2019).The most common indications for a serum protein electrophoresis test are to diagnose or monitor multiple myeloma, a monoclonal gammopathy of uncertain significance (MGUS), or further investigate a discrepancy between a low albumin and a relatively high total protein.
Unexplained bone pain, anemia, proteinuria, chronic kidney disease, and hypercalcemia are also signs of multiple myeloma, and indications for SPE. 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 applied to either an acetate membrane soaked in a liquid buffer, or to a buffered agarosegel matrix, 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 1 and 2 globulins, and gamma globulins(Jenkins, 2019).
Electrophoresis is a method of separating proteins based on their physical properties. Serum is placed on a specific medium, and a charge is applied. The net charge (positive or negative) and the size and shape of the protein commonly are used in differentiating various serum proteins.
Several subsets of serum protein electrophoresis are available. The names of these subsets are based on the method that is used to separate and differentiate the various serum components. In zone electrophoresis, for example, different protein subtypes are placed in separate physical locations on a gel made from agar, cellulose, or other plant material.The proteins are stained, and their densities are calculated electronically to provide graphical data on the absolute and relative amounts of the various proteins. Further separation of protein subtypes is achieved by staining with an immunologically active agent, which results in immunofluorescence and immunofixation (Harris et al., 2012)
ACETATE OR GEL ELECTROPHORESIS
Proteins are separated by both electrical forces and electroendoosmostic forces. The net charge on a protein is based on the sum charge of its amino acids, and the pH of the buffer. Proteins are applied to a solid matrix such as an agarose gel, or a cellulose acetate membrane in a liquid buffer, and electric current is applied. Proteins with a negative charge will migrate towards the positively charged anode. Albumin has the most negative charge, and will migrate furthest towards the anode. Endoosmotic flow is the movement of liquid towards the cathode, which causes proteins with a weaker charge to move backwards from the application site. Gamma proteins are primarily separated by endoosmotic forces (Kaplan and Savor, 2015)
CAPILLARY ELECTROPHORESIS
In capillary electrophoresis, there is no solid matrix. Proteins are separated primarily by strong electroendosmotic forces. The sample is injected into a capillary with a negative surface charge. A high current is applied, and negatively charged proteins such as albumin try to move towards the anode. Liquid buffer flows towards the cathode, and drags proteins with a weaker charge
COMPONENTS OF SERUM PROTEIN ELECTROPHORESIS
The pattern of serum protein electrophoresis results depends on the fractions of two major types of protein: albumin and globulins. Albumin, the major protein component of serum, is produced by the liver under normal physiologic conditions. Globulins comprise a much smaller fraction of the total serum protein content. The subsets of these proteins and their relative quantity are the primary focus of the interpretation of serum protein electrophoresis (Keren, 2013).
Albumin, the largest peak, lies closest to the positive electrode. The next five components (globulins) are labeled alpha1, alpha2, beta1, beta2, and gamma. The peaks for these components lie toward the negative electrode, with the gamma peak being closest to that electrode. Figure 1shows a typical normal pattern for the distribution of proteins as determined by serum protein electrophoresis.
a. ALBUMIN
The albumin band represents the largest protein component of human serum. The albumin level is decreased under circumstances in which there is less production of the protein by the liver or in which there is increased loss or degradation of this protein. Malnutrition, significant liver disease, renal loss (e.g., in nephrotic syndrome), hormone therapy, and pregnancy may account for a low albumin level. Burns also may result in a low albumin level. Levels of albumin are increased in patients with a relative reduction in serum water (e.g., dehydration).
b. ALPHA FRACTION
Moving toward the negative portion of the gel (i.e., the negative electrode), the next peaks involve the alpha1 and alpha2 components. The alpha1-protein fraction is comprised of alpha1-antitrypsin, thyroid-binding globulin, and transcortin. Malignancy and acute inflammation (resulting from acute-phase reactants) can increase the alpha1-protein band. A decreased alpha1-protein band may occur because of alpha1-antitrypsin deficiency or decreased production of the globulin as a result of liver disease. Ceruloplasmin, alpha2-macroglobulin, and haptoglobin contribute to the alpha2-protein band. The alpha2 component is increased as an acute-phase reactant.
c. BETA FRACTION
The beta fraction has two peaks labeled beta1 and beta2. Beta1 is composed mostly of transferrin, and beta2 contains beta-lipoprotein. IgA, IgM, and sometimes IgG, along with complement proteins, also can be identified in the beta fraction.
d. GAMMA FRACTION
Much of the clinical interest is focused on the gamma region of the serum protein spectrum because immunoglobulins migrate to this region. It should be noted that immunoglobulins often can be found throughout the electrophoretic spectrum. C-reactive protein (CRP) is located in the area between the beta and gamma components.(Keren, 2013).
INDICATIONS
Serum protein electrophoresis commonly is performed when multiple myeloma is suspected. The examination also should be considered in other “red flag” situations.
If the examination is normal but multiple myeloma, Waldenström’smacroglobulinemia, primary amyloidosis, or a related disorder still is suspected, immunofixation also should be performed because this technique may be more sensitive in identifying a small monoclonal (M) protein (Keren, 2013).
Interpretation of Results
Plasma protein levels display reasonably predictable changes in response to acute inflammation, malignancy, trauma, necrosis, infarction, burns, and chemical injury. This so-called “acute-reaction protein pattern” involves increases in fibrinogen, alpha1-antitrypsin, haptoglobin, ceruloplasmin, CRP, the C3 portion of complement, and alpha1 acid glycoprotein. Often, there are associated decreases in the albumin and transferrin levels.Table below lists characteristic patterns of acute-reaction proteins found on serum protein electrophoresis, along with associated conditions or disorders.
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