Albumin Albumin is the major fraction in a normal SPEP. A fall of 30% is necessary before the decrease shows on electrophoresis. Usually a single band is seen.
Heterozygous individuals may produce
bisalbuminemia – two equally staining bands, the product of two genes. Some variants give rise to a wide band or two bands of unequal intensity but
none of these variants is associated with disease. Increased anodic mobility results from the binding of
bilirubin, nonesterified
fatty acids,
penicillin and
acetylsalicylic acid, and occasionally from tryptic digestion in acute
pancreatitis. Absence of albumin, known as
analbuminaemia, is rare. A decreased level of albumin, however, is common in many diseases, including
liver disease,
malnutrition, malabsorption, protein-losing nephropathy and enteropathy.
Albumin – alpha-1 interzone Even staining in this zone is due to alpha-1 lipoprotein (
high density lipoprotein – HDL). Decrease occurs in severe inflammation, acute
hepatitis, and
cirrhosis. Also,
nephrotic syndrome can lead to decrease in albumin level; due to its loss in the urine through a damaged leaky
glomerulus. An increase appears in severe alcoholics and in women during pregnancy and in puberty. The high levels of
AFP that may occur in
hepatocellular carcinoma may result in a sharp band between the albumin and the alpha-1 zone.
Alpha-1 zone Orosomucoid and
antitrypsin migrate together but orosomucoid stains poorly so
alpha 1 antitrypsin (AAT) constitutes most of the alpha-1 band. Alpha-1 antitrypsin has an SG group and thiol compounds may be bound to the protein altering their mobility. A decreased band is seen in the deficiency state. It is decreased in the
nephrotic syndrome and absence could indicate possible alpha 1-antitrypsin deficiency. This eventually leads to
emphysema from unregulated neutrophil elastase activity in the lung tissue. The alpha-1 fraction does not disappear in alpha 1-antitrypsin deficiency, however, because other proteins, including alpha-
lipoprotein and orosomucoid, also migrate there. As a positive acute phase reactant, AAT is increased in acute inflammation.
Bence Jones protein may bind to and retard the alpha-1 band.
Alpha-1 – alpha-2 interzone Two faint bands may be seen representing
alpha 1-antichymotrypsin and
vitamin D binding protein. These bands fuse and intensify in early inflammation due to an increase in alpha 1-antichymotrypsin, an
acute phase protein.
Alpha-2 zone This zone consists principally of
alpha-2 macroglobulin (AMG or A2M) and
haptoglobin. There are typically low levels in
haemolytic anaemia (haptoglobin is a suicide molecule which binds with free
haemoglobin released from
red blood cells and these complexes are rapidly removed by
phagocytes). Haptoglobin is raised as part of the acute phase response, resulting in a typical elevation in the alpha-2 zone during inflammation. A normal alpha-2 and an elevated alpha-1 zone is a typical pattern in hepatic
metastasis and cirrhosis. Haptoglobin/haemoglobin complexes migrate more cathodally than haptoglobin as seen in the alpha-2 – beta interzone. This is typically seen as a broadening of the alpha-2 zone. Alpha-2 macroglobulin may be elevated in children and the elderly. This is seen as a sharp front to the alpha-2 band. AMG is markedly raised (10-fold increase or greater) in association with glomerular protein loss, as in
nephrotic syndrome. Due to its large size, AMG cannot pass through glomeruli, while other lower-molecular weight proteins are lost. Enhanced synthesis of AMG accounts for its absolute increase in nephrotic syndrome. Increased AMG is also noted in rats with no albumin indicating that this is a response to low albumin rather than nephrotic syndrome itself AMG is mildly elevated early in the course of
diabetic nephropathy.
Alpha-2 - beta interzone Cold insoluble globulin forms a band here which is not seen in
plasma because it is precipitated by
heparin. There are low levels in inflammation and high levels in pregnancy. Beta lipoprotein forms an irregular
crenated band in this zone. High levels are seen in type II
hypercholesterolaemia,
hypertriglyceridemia, and in the nephrotic syndrome.
Beta zone Transferrin and
beta-lipoprotein (
LDL) comprises the beta-1. Increased beta-1 protein due to the increased level of free transferrin is typical of
iron deficiency anemia,
pregnancy, and
oestrogen therapy. Increased beta-1 protein due to LDL elevation occurs in
hypercholesterolemia. Decreased beta-1 protein occurs in acute or chronic inflammation. Beta-2 comprises C3 (
complement protein 3). It is raised in the acute phase response. Depression of C3 occurs in autoimmune disorders as the complement system is activated and the C3 becomes bound to immune complexes and removed from serum. Fibrinogen, a beta-2 protein, is found in normal plasma but absent in normal serum. Occasionally, blood drawn from heparinized patients does not fully clot, resulting in a visible fibrinogen band between the beta and gamma globulins.
Beta-gamma interzone C-reactive protein is found in between the beta and gamma zones producing beta/gamma fusion.
IgA has the most anodal mobility and typically 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).
Fibrinogen from
plasma samples will be seen in the beta gamma region. Fibrinogen, a beta-2 protein, is found in normal plasma but absent in normal serum. Occasionally, blood drawn from heparinized patients does not fully clot, resulting in a visible fibrinogen band between the beta and gamma globulins.
Gamma zone The
immunoglobulins or
antibodies are generally the only proteins present in the normal gamma region. Of note, any protein migrating in the gamma region will be stained and appear on the gel, which may include protein contaminants, artifacts, or certain medications. Depending on whether an agarose or capillary method is used, interferences vary. Immunoglobulins consist of heavy chains (μ, δ, γ, α, and ε) and light chains (κ and λ). A normal gamma zone should appear as a smooth 'blush', or smear, with no asymmetry or sharp peaks. The gamma globulins may be elevated (
hypergammaglobulinemia), decreased (
hypogammaglobulinaemia), or have an abnormal peak or peaks. Note that immunoglobulins may also be found in other zones; IgA typically migrates in the beta-gamma zone, and in particular, pathogenic immunoglobulins may migrate anywhere, including the alpha regions. Hypogammaglobulinaemia is easily identifiable as a "slump" or decrease in the gamma zone. It is normal in infants. It is found in patients with
X-linked agammaglobulinemia. IgA deficiency occurs in 1:500 of the population, as is suggested by a pallor in the gamma zone. Of note, hypogammaglobulinema may be seen in the context of MGUS or multiple myeloma. If the gamma zone shows an increase the first step in interpretation is to establish if the region is narrow or wide. A broad "swell-like" manner (wide) indicates polyclonal immunoglobulin production. If it is elevated in an asymmetric manner or with one or more peaks or narrow "spikes" it could indicate clonal production of one or more immunoglobulins, Polyclonal gammopathy is indicated by a "swell-like" elevation in the gamma zone, which typically indicates a non-neoplastic condition (although is not exclusive to non-neoplastic conditions). The most common causes of polyclonal hypergammaglobulinaemia detected by electrophoresis are severe
infection, chronic liver disease, rheumatoid arthritis, systemic
lupus erythematosus and other connective tissue diseases. A narrow spike is suggestive of a monoclonal gammopathy, also known as a restricted band, or "M-spike". To confirm that the restricted band is an immunoglobulin, follow up testing with
immunofixation, or immunodisplacement/immunosubtraction (capillary methods) is performed. Therapeutic
monoclonal antibodies (mAb), also migrate in this region and may be misinterpreted as a monoclonal gammopathy, and may also be identified by immunofixation or immunodisplacement/immunosubtraction as they are structurally comparable to human immunoglobulins. The most common cause of a restricted band is an MGUS (monoclonal gammopathy of uncertain significance), which, although a necessary precursor, only rarely progresses to multiple myeloma. (On average, 1%/year.) Typically, a monoclonal gammopathy is malignant or clonal in origin,
Myeloma being the most common cause of
IgA and
IgG spikes. chronic lymphatic leukaemia and
lymphosarcoma are not uncommon and usually give rise to
IgM paraproteins. Note that up to 8% of healthy geriatric patients may have a monoclonal spike.
Waldenström's macroglobulinaemia (IgM),
monoclonal gammopathy of undetermined significance (MGUS), amyloidosis, plasma cell leukemia and solitary plasmacytomas also produce an M-spike. Oligoclonal gammopathy is indicated by one or more discrete clones.
Lysozyme may be seen as a band cathodal to gamma in myelomonocytic
leukaemia in which it is released from
tumour cells. ==References==