Electrophoresis is still the most commonly used screening test for serum protein abnormalities except for measurement of albumin only. Before electrophoresis was readily available, the albumin/globulin ratio was widely used; this was determined with a chemical method and should no longer be ordered, since electrophoresis not only provides the same information but also pinpoints areas where globulin abnormalities may be found. Various electrophoretic methods produce some differences within the same basic framework of results due to differences in technical factors and the type of electrophoretic migrating field material used. Some of the standard materials include filter paper, cellulose acetate film, agarose gel, and polyacrylamide gel. Each method has advantages and disadvantages in terms of laboratory ease of performance, cost, or sensitivity to certain protein fractions. However, all are capable of identifying the areas where major serum protein shifts take place, which sometimes have as much diagnostic importance as the shifts themselves.

Serum protein electrophoresis ordinarily displays bands corresponding to albumin, alpha-1 and alpha-2 globulins, beta globulins, and gamma globulins. A special instrument (densitometer) translates the quantity of protein or density of these bands into a linear pattern, which is a rough visual approximation of the amount of substance present. The main problem with this method is the difficulty in separating some of the serum components. It has been found that by using potato starch in a gel-like state, the separation of some of these fractions can be sharpened, especially the separation of some of the abnormal hemoglobins. Polyacrylamide gel has also been used. However, these procedures are technically somewhat more difficult than the other techniques, so that filter paper, cellulose acetate, and agarose gel remain the routine clinical laboratory methods of choice. On filter paper, cellulose acetate, or agarose, the alpha-2 fraction migrates faster toward the anode than the beta fraction. On starch or polyacrylamide gel, the reverse occurs.

Proteins visualized by serum protein electrophoresis: acute reaction proteins. Certain reasonably predictable changes take place in plasma protein levels in response to acute illness, including acute inflammation, trauma, necrosis, infarction, burns, and chemical injury. The same changes may occur in focal episodes associated with malignant tumors (possibly due to infarction of a portion of the tumor). The acute reaction protein pattern has also been called “acute inflammatory response pattern,” “acute stress pattern,” and “acute-phase protein pattern.” The protein changes involved are increases in fibrinogen, alpha-1 antitrypsin, haptoglobin, ceruloplasmin, C-reactive protein (CRP), C3 portion of complement, and alpha-1 acid glycoprotein (orosomucoid) levels. There frequently is an associated decrease in albumin and transferrin levels. Of those proteins that are increased, the greatest effect is produced by haptoglobin, alpha-1 antitrypsin, CRP, and fibrinogen. The CRP level increase begins approximately 4-6 hours after onset of the acute episode, with alterations of the other proteins occurring 12-36 hours after onset. CRP is located in the beta-gamma interface area on electrophoresis, and a distinct peak is not usually seen. Fibrinogen is normally not present in serum (unless the blood has not completely clotted), so ordinarily it is not detected on serum electrophoresis. Haptoglobin migrates in the alpha-2 region, with the result that alpha-2 elevation is the most common abnormality associated with acute reaction. An alpha-1 elevation due to alpha-1 antitrypsin increase is seen less frequently but can be present. Albumin is often decreased under acute reaction conditions, presumably due to decreased liver synthesis, for which there often is no good explanation. Albumin is not always decreased; due to the wide reference range, a substantial reduction could occur in a person whose normal level is toward the upper end of the reference range, with the final level remaining within the lower reference range despite the reduction. A decrease in transferrin levels is usually not seen on electrophoresis but is manifested by a decrease in the total iron-binding capacity. In summary, the typical electrophoretic change of acute reaction is increased alpha-2 globulin, frequently associated with decreased albumin and sometimes with increased alpha-1 globulin. Acute reaction frequently is accompanied by a polymorphonuclear leukocytosis and usually by an increase in CRP test values (especially when using immunoassay methods) and an increased erythrocyte sedimentation rate (ESR).

Serum albumin. Elevation of the serum albumin level is very unusual other than in dehydration. Most changes involve diminution, although the normal range is somewhat large, and small decreases are thus hidden unless the individual patient’s normal levels are known. In pregnancy, albumin levels decrease progressively until delivery and do not return to normal until about 8 weeks post partum. In infants, adult levels are reached at about age 1 year. Thereafter, serum protein levels are relatively stable except for a gradual decrease after age 70. Malnutrition leads to decreased albumin levels, presumably from lack of essential amino acids, but also possibly from impaired liver manufacture and unknown causes Impaired synthesis may itself be a cause of decreased albumin levels, since it is found in most forms of clinical liver disease, especially cirrhosis. In chronic cachectic or wasting diseases, such as tuberculosis or carcinoma, the albumin level is often decreased, but it is not clear whether this is due to impaired synthesis or to other factors. Chronic infections seem to have much the same effect as the cachectic diseases.

Serum albumin may be directly lost from the bloodstream by hemorrhage, burns, exudates, or leakage into the gastrointestinal (GI) tract in various protein-losing enteropathies. In the nephrotic syndrome there is a marked decrease in the serum albumin level from direct loss into the urine. Albumin frequently decreases rather quickly in many severe acute illnesses or injuries, beginning at about 12-36 hours, with the average maximal albumin decrease being reached in about 5 days. This is part of the acute reaction pattern described earlier and seems to have a different mechanism from hypoalbuminemia due to protein loss or to malnutrition. Finally, there is a rare genetic cause for low serum albumin levels known as “familial idiopathic dysproteinemia,” in which the albumin level is greatly decreased while all the globulin fractions are elevated and seem to take over most of the functions of albumin.

Alpha-1 globulins. The alpha-1 globulin area on electrophoresis is almost 90% alpha-1 antitrypsin. The other 10% includes alpha-1 acid glycoprotein, alpha-fetoprotein, and certain carrier proteins such as cortisol-binding protein (transcortin) and thyroxine-binding globulin (which is actually located more toward the area between alpha-1 and alpha-2). Alpha-1 globulin is increased to some extent (usually not great) in pregnancy and by estrogen administration and in some patients with the acute reaction protein pattern. Alpha-1 globulin is absent or nearly so in alpha-1 antitrypsin deficiency, a hereditary disorder that predisposes to development of emphysema. Serum protein electrophoresis detects homozygous alpha-1 antitrypsin deficiency but frequently displays a normal alpha-1 peak in those who are heterozygous. Immunoassay is needed to detect heterozygotes or to confirm electrophoretic findings.

Alpha-2 globulins. Alpha-2 globulins include haptoglobin, alpha-2 macroglobulin, and ceruloplasmin. This electrophoretic area is seldom depressed; diminution of one component is usually masked by the other components within the reference range. Haptoglobin values are decreased in severe liver disease, in patients on estrogen therapy, in megaloblastic anemia, and also whenever free hemoglobin appears in the blood, as occurs in red blood cell (RBC) hemolysis or even from resorption of a large hematoma located outside the vascular system. Haptoglobin levels are increased by adrenocorticosteroid therapy. Ceruloplasmin levels are decreased in Wilson’s disease, malnutrition, nephrotic syndrome, and protein-losing enteropathy. Ceruloplasmin levels are increased by estrogen therapy. Both haptoglobin and ceruloplasmin levels are increased, resulting in alpha-2 elevation, in the many disorders that produce the acute reaction pattern.

In subacute and severe chronic illnesses the acute reaction pattern may exist in a lesser degree or may disappear. The alpha-2 increase usually diminishes and may return to normal. Albumin levels may return to normal, but an albumin decrease frequently persists or may even become more pronounced. Gamma-globulin levels may begin to increase.

There are certain diseases other than acute injury that often produce alpha-2 increase. In the nephrotic syndrome there is classically a marked alpha-2 peak, which may sometimes be accompanied by a beta-globulin elevation. In addition there is greatly decreased albumin. In hyperthyroidism, far-advanced diabetes, and adrenal insufficiency, there is reportedly a slightly to moderately elevated alpha-2 globulin level in some cases.

Beta globulins. The beta-globulin zone contains transferrin, beta-lipoprotein, and several components of complement.

A decrease in the beta-globulin level is not very common. Transferrin is frequently decreased in protein malnutrition. Beta globulin increase may occur in many conditions. Probably the most common source is a nonfasting specimen. An increase in transferrin levels produced by chronic iron deficiency anemia, pregnancy in the third trimester, and free hemoglobin in serum, or fibrinogen from incompletely clotted blood may each produce a spikelike peak in the beta region that simulates a monoclonal peak. In conditions in which serum cholesterol levels are elevated, the beta-globulin levels are also likely to be increased; these include hypothyroidism, biliary cirrhosis, nephrosis, and some cases of diabetes. In liver disease, there is some variability. The beta-globulin level is usually, but not always, elevated in obstructive jaundice. It may be elevated to some extent in many cases of hepatitis but not as often as in obstructive jaundice. It is often elevated in cirrhosis; when so, it is often partially incorporated into the gamma globulin and occasionally does not even appear as a separate peak. This will be discussed later. Finally, beta elevation may occasionally be seen in certain other diseases, including malignant hypertension, Cushing’s disease, polyarteritis nodosa, and sometimes carcinoma. These changes are probably due to increase in complement. A double peak in the beta area is a frequent and normal finding when the cellulose acetate method is used. Electrophoresis on polyacrylamide gel produces reversal of the alpha-2 and beta area positions compared to paper or cellulose acetate electrophoresis (i.e., the beta area on paper becomes alpha-2 on polyacrylamide gel).

Gamma globulins. The gamma region is predominantly composed of antibodies of the IgG type. Immunoglobulin A, IgM, IgD, and IgE antibodies underlie the beta-gamma junction area. The gamma-globulin zone is decreased in hypogammagobulinemia and agammaglobulinemia, which may be either primary or secondary. The secondary type sometimes may be found in patients on long-term steroid treatment, in the nephrotic syndrome, in occasional patients with overwhelming infection, and in a moderate number of patients with chronic lymphocytic leukemia, lymphocytic lymphoma, or multiple myeloma of the light chain type.

Many diseases produce an increase in the gamma-globulin level. Many types of infections are followed by an increased gamma-globulin level that reflects antibody production, although the increase is often not sufficient to demonstrate a clear-cut elevation above reference range, especially if the infection is mild or acute. Chronic infections typically produce antibody responses that are prolonged and substantial enough to increase gamma-globulin values above reference limits. Granulomatous diseases such as tuberculosis, sarcoidosis, lymphogranuloma venereum, and tertiary syphilis are also chronic diseases and frequently result in marked gamma increase by the time they become well established. Rheumatoid-collagen diseases, notably rheumatoid arthritis and lupus erythematosis, have electrophoretic gamma values that range from normal to considerably increased. Gamma levels are usually elevated while the disease is active if activity has persisted for several months. Gamma-globulin levels may be increased in some patients with Hodgkin’s disease, malignant lymphoma, and chronic lymphocytic leukemia, although in other patients they may be decreased or normal. Multiple myeloma and Waldenstrцm’s macroglobulinemia characteristically demonstrate a homogeneous spikelike peak in a focal region of the gamma area, which may or may not result in the value for the total gamma area being increased. Liver diseases form a substantial group of etiologies for gamma elevation. In hepatitis there is classically a relatively mild separate increase in both beta- and gamma-globulin levels with a decrease in albumin level, but this does not always occur. About 90% of patients with well-established cirrhosis show some degree of gamma elevation. The gamma elevation is of considerable degree in about 30% of patients and of slight to moderate degree in about 60%. There is no electrophoretic gamma elevation in about 10% of patients with histologically well-established cirrhosis. The most suggestive pattern is a broad-based gamma-globulin elevation plus a fusion of beta and gamma globulin without the usual separation of the two peaks (“beta-gamma bridging”). However, only about 20% of cirrhotics display complete beta-gamma fusion with about 33% more showing partial fusion. In obstructive jaundice, alpha-2, beta, and gamma levels may all become elevated to some degree.