 Serum protein electrophoresis is an easy method of separating serum proteins based on their net charge, size, and shape.  A small amount of serum is placed on a specific medium (such as agarose) and an electrical charge is applied.  The proteins then migrate across the medium in a characteristic manner, due to the net charge and size and shape of the protein.  In routine serum protein electrophoresis, the protein will separate into five main components, identified as albumin and the globulins (alpha1, alpha2, beta and gamma).

 Approximately 60% of the total protein in the serum is albumin, and represents the largest peak that lies closest to the positive electrode, while the remaining fractions are composed mainly of globulins  Globulins comprise a much smaller fraction of the total serum protein but represent the primary focus of interpretation of serum protein electrophoresis. monoclonal gammopathies  The γ region contains the largest portion of globulins, therefore monoclonal gammopathies are most frequently encountered in the electrophoresis

 Albumin represents the highest peak in serum protein electrophoresis, usually seen as a single, tall band.  Occasionally, two equally staining bands, referred to as bisalbuminemia or a widely staining band may be seen, which both represent normal variations.  Common conditions associated with decreased albumin include malnutrition, cirrhosis and nephrotic syndrome.  Dehydration, on the other hand, causes a high albumin.  Absence of albumin, known as analbuminaemia, is rare

 The alpha-1 fraction includes α -1 antitrypsin, transcortin, and thyroid- binding globulin.  α -2 fraction is comprised of ceruloplasmin, α -2macroglobulin, and haptoglobin.  Both alpha-1 and 2 represent the acute phase reactants; hence, malignancy, infection or any inflammatory condition can cause their elevation.

 A relative increase in α -2 fraction may be seen in nephrotic syndrome due to the relative larger size of the proteins and the inability to pass through the glomeruli.  A decrease in α -1 component may be seen in α -1 antitrypsin deficiency  A decrease in alpha-2 component may be seen in hemolytic anemia due to decreased haptoglobin levels.

 The beta zone consist of β -1 and β -2 but is often represented a graphically as a single band.  β -1 consists mostly of transferrin, and is increased in conditions such as iron- deficiency anemia, pregnancy and estrogen therapy. B-lipoprotein and C3 complement are included in the β -2 component.  IgA has an anodal mobility and migrates in the region between the beta and gamma zones also causing a beta/gamma fusion in patients with cirrhosis, respiratory infection, skin disease, or rheumatoid arthritis (increased IgA).

 Immunoglobulins mainly comprise this area including IgG, IgA, IgM, IgD and IgE, but note that Igs may be found in the α and β zones.  If the γ zone shows an increase (or spike), the first step in interpretation is to establish if the region is narrow or wide.  If it is elevated in a single narrow "spike-like" manner it could indicate monoclonal production of a single immunoglobulin (monoclonal gammopathy), while a broad "swell- like" manner (wide) indicates polyclonal immunoglobulin production.

 Various inflammatory, autoimmune and hematologic, and non- hematologic diseases are associated with an increased gamma peak  Agammaglobulinemia and hypogammaglobulinemia syndromes such as IgA deficiency are associated with a decrease in this area.

 Standard SPE separates the protein into five distinct bands but, by modifying the electrophoretic parameters, proteins can be further separated into as many as 12 bands.  The modification, known as HRE, uses a higher voltage coupled with a cooling system in the electrophoretic apparatus and a more concentrated buffer.

 High (or low) total serum globulin or immunoglobulin  Extremely high percentage of lymphocytes  Unexplained anaemia (multiple myeloma is a recognised cause of non-iron deficiency anaemia) or other persisting cytopaenias for which there is no other explanation  Unexplained high ESR (>50) with a normal CRP  Unexplained hypercalcaemia or renal impairment  Red cell rouleaux formations noted on the peripheral blood smear  Presence of urine free light chains (Bence-Jones proteinuria)

 The specimen most often used to determine the total protein is serum rather than plasma.  Container : Red-top tube, serum separator tube (SST)  A fasting specimen is not needed.  Interferences in some of the methods occur in the presence of lipemia; hemolysis falsely elevates the total protein result because of the release of RBC proteins into the serum.

 When an abnormality is found in the total protein or albumin, an electrophoresis is usually performed.  In the standard method for serum protein electrophoresis (SPE) : 1. serum samples are applied close to the cathode end of a support medium that is saturated with an alkaline buffer (pH 8.6). 2. The support medium is connected to two electrodes and a current is passed through the medium to separate the proteins. 3. All major serum proteins carry a net negative charge at pH 8.6 and migrate toward the anode.

 Many scanning densitometers compute the area under the absorbance curve for each band and the percentage of total dye that appears in each fraction.  The concentration is then calculated as a percentage of the total protein that was determined by one of the protein methods, such as the biuret procedure.  For example: n Spike is 60% of total protein n Total protein=12 grams/deciliter (g/dL) n Spike level=7.2 g/dL (60% of 12)

 A reference serum control is processed with each electrophoretic run  Reference values for each fraction are as follows: 1. Albumin : 53–65% of the total protein (3.5–5.0 g/dL) 2. α-1 : 2.5–5% (0.1–0.3 g/dL) 3. α-2 : 7–13% (0.6–1.0 g/dL) 4. β : 8–14% (0.7–1.1 g/dL) 5. γ : 12–22% (0.8–1.6 g/dL)

 The great advantage of electrophoresis compared with the quantitation of specific proteins is the overview it provides.  The electrophoretic pattern can give information about the relative increases and decreases within the protein population, as well as information about the homogeneity of a fraction.

Albumin decreased, alpha1,alpha2 and beta increased

Albumin decreased and gamma globuline increased

 Probably the most significant finding from an electrophoretic pattern

Polyclonal Immunoglobulin disease