serum proteins
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Serum protein electrophoresis (SPEP or SPE) is a laboratory test that examines specific proteins in the blood called
globulins The globulins are a family of globular proteins that have higher molecular weights than albumins and are insoluble in pure water but dissolve in dilute salt solutions. Some globulins are produced in the liver, while others are made by the immune ...
. 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 Proteinuria is the presence of excess proteins in the urine. In healthy persons, urine contains very little protein; an excess is suggestive of illness. Excess protein in the urine often causes the urine to become foamy (although this symptom ma ...
, chronic kidney disease, and hypercalcemia are also signs of multiple myeloma, and indications for SPE.
Blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood in the cir ...
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 agarose
gel A gel is a semi-solid that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state, although the liquid phase may still dif ...
matrix, or into liquid in a
capillary A capillary is a small blood vessel from 5 to 10 micrometres (μm) in diameter. Capillaries are composed of only the tunica intima, consisting of a thin wall of simple squamous endothelial cells. They are the smallest blood vessels in the bod ...
tube, and exposed to an electric current to separate the serum protein components into five major fractions by size and electrical charge:
serum albumin Serum albumin, often referred to simply as blood albumin, is an albumin (a type of globular protein) found in vertebrate blood. Human serum albumin is encoded by the ''ALB'' gene. Other mammalian forms, such as bovine serum albumin, are chemica ...
, alpha-1 globulins, alpha-2 globulins, beta 1 and 2 globulins, and gamma globulins.


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.


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.


Serum protein fractions


Albumin

Albumin Albumin is a family of globular proteins, the most common of which are the serum albumins. All the proteins of the albumin family are water-soluble, moderately soluble in concentrated salt solutions, and experience heat denaturation. Albumins ...
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 Zygosity (the noun, zygote, is from the Greek "yoked," from "yoke") () is the degree to which both copies of a chromosome or gene have the same genetic sequence. In other words, it is the degree of similarity of the alleles in an organism. Mo ...
individuals may produce
bisalbuminemia Bisalbuminemia is the, sometimes inherited, condition of having two types of serum albumin Serum albumin, often referred to simply as blood albumin, is an albumin (a type of globular protein) found in vertebrate blood. Human serum albumin is ...
– 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 In chemistry, particularly in biochemistry, a fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated and unsaturated compounds#Organic chemistry, saturated or unsaturated. Most naturally occurring fatty acids have an B ...
, penicillin and
acetylsalicylic acid Aspirin, also known as acetylsalicylic acid (ASA), is a nonsteroidal anti-inflammatory drug (NSAID) used to reduce pain, fever, and/or inflammation, and as an antithrombotic. Specific inflammatory conditions which aspirin is used to treat ...
, and occasionally from tryptic digestion in acute
pancreatitis Pancreatitis is a condition characterized by inflammation of the pancreas. The pancreas is a large organ behind the stomach that produces digestive enzymes and a number of hormones. There are two main types: acute pancreatitis, and chronic pancr ...
. Absence of albumin, known as
analbuminaemia Analbuminaemia or analbuminemia is a genetically inherited metabolic defect characterised by an impaired synthesis of serum albumin. Although albumin Albumin is a family of globular proteins, the most common of which are the serum albumins. ...
, is rare. A decreased level of albumin, however, is common in many diseases, including
liver disease Liver disease, or hepatic disease, is any of many diseases of the liver. If long-lasting it is termed chronic liver disease. Although the diseases differ in detail, liver diseases often have features in common. Signs and symptoms Some of the si ...
,
malnutrition Malnutrition occurs when an organism gets too few or too many nutrients, resulting in health problems. Specifically, it is "a deficiency, excess, or imbalance of energy, protein and other nutrients" which adversely affects the body's tissues ...
, malabsorption, protein-losing nephropathy and enteropathy.


Albumin – alpha-1 interzone

Even staining in this zone is due to alpha-1 lipoprotein (
high density lipoprotein High-density lipoprotein (HDL) is one of the five major groups of lipoproteins. Lipoproteins are complex particles composed of multiple proteins which transport all fat molecules (lipids) around the body within the water outside cells. They are t ...
– HDL). Decrease occurs in severe inflammation, acute
hepatitis Hepatitis is inflammation of the liver tissue. Some people or animals with hepatitis have no symptoms, whereas others develop yellow discoloration of the skin and whites of the eyes ( jaundice), poor appetite, vomiting, tiredness, abdominal ...
, and
cirrhosis Cirrhosis, also known as liver cirrhosis or hepatic cirrhosis, and end-stage liver disease, is the impaired liver function caused by the formation of scar tissue known as fibrosis due to damage caused by liver disease. Damage causes tissue rep ...
. Also, nephrotic syndrome can lead to decrease in albumin level; due to its loss in the urine through a damaged leaky
glomerulus ''Glomerulus'' () is a common term used in anatomy to describe globular structures of entwined vessels, fibers, or neurons. ''Glomerulus'' is the diminutive of the Latin ''glomus'', meaning "ball of yarn". ''Glomerulus'' may refer to: * the filter ...
. 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 Orosomucoid (ORM) or alpha-1-acid glycoprotein (''α1AGp'', ''AGP'' or ''AAG'') is an acute phase protein found in plasma. It is an alpha-globulin glycoprotein and is modulated by two polymorphic genes. It is synthesized primarily in hepatoc ...
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 A lipoprotein is a biochemical assembly whose primary function is to transport hydrophobic lipid (also known as fat) molecules in water, as in blood plasma or other extracellular fluids. They consist of a triglyceride and cholesterol center, su ...
and orosomucoid, also migrate there. As a positive acute phase reactant, AAT is increased in acute inflammation.
Bence Jones protein Bence Jones protein is a monoclonal globulin protein or immunoglobulin light chain found in the urine, with a molecular weight of 22–24 kDa. Detection of Bence Jones protein may be suggestive of multiple myeloma or Waldenström's macroglobuli ...
may bind to and retard the alpha-1 band.


Alpha-1 – alpha-2 interzone

Two faint bands may be seen representing
alpha 1-antichymotrypsin Alpha 1-antichymotrypsin (symbol α1AC, A1AC, or a1ACT) is an alpha globulin glycoprotein that is a member of the serpin superfamily. In humans, it is encoded by the ''SERPINA3'' gene. Function Alpha 1-antichymotrypsin inhibits the activity o ...
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 Haptoglobin (abbreviated as Hp) is the protein that in humans is encoded by the ''HP'' gene. In blood plasma, haptoglobin binds with high affinity to ''free'' hemoglobin released from erythrocytes, and thereby inhibits its deleterious oxidative ...
. There are typically low levels in
haemolytic anaemia Hemolytic anemia or haemolytic anaemia is a form of anemia due to hemolysis, the abnormal breakdown of red blood cells (RBCs), either in the blood vessels (intravascular hemolysis) or elsewhere in the human body (extravascular). This most commonly ...
(haptoglobin is a suicide molecule which binds with free
haemoglobin Hemoglobin (haemoglobin BrE) (from the Greek word αἷμα, ''haîma'' 'blood' + Latin ''globus'' 'ball, sphere' + ''-in'') (), abbreviated Hb or Hgb, is the iron-containing oxygen-transport metalloprotein present in red blood cells (erythrocyte ...
released from red blood cells and these complexes are rapidly removed by
phagocytes Phagocytes are cells that protect the body by ingesting harmful foreign particles, bacteria, and dead or dying cells. Their name comes from the Greek ', "to eat" or "devour", and "-cyte", the suffix in biology denoting "cell", from the Greek '' ...
). 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 Metastasis is a pathogenic agent's spread from an initial or primary site to a different or secondary site within the host's body; the term is typically used when referring to metastasis by a cancerous tumor. The newly pathological sites, then ...
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 Diabetic nephropathy, also known as diabetic kidney disease, is the chronic loss of kidney function occurring in those with diabetes mellitus. Diabetic nephropathy is the leading causes of chronic kidney disease (CKD) and end-stage renal disease ...
.


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 Hypercholesterolemia, also called high cholesterol, is the presence of high levels of cholesterol in the blood. It is a form of hyperlipidemia (high levels of lipids in the blood), hyperlipoproteinemia (high levels of lipoproteins in the blood), ...
,
hypertriglyceridemia Hypertriglyceridemia is the presence of high amounts of triglycerides in the blood. Triglycerides are the most abundant fatty molecule in most organisms. Hypertriglyceridemia occurs in various physiologic conditions and in various diseases, and ...
, and in the nephrotic syndrome.


Beta zone

Transferrin Transferrins are glycoproteins found in vertebrates which bind to and consequently mediate the transport of iron (Fe) through blood plasma. They are produced in the liver and contain binding sites for two Fe3+ ions. Human transferrin is encode ...
and beta-lipoprotein (
LDL Low-density lipoprotein (LDL) is one of the five major groups of lipoprotein that transport all fat molecules around the body in extracellular water. These groups, from least dense to most dense, are chylomicrons (aka ULDL by the overall densit ...
) comprises the beta-1. Increased beta-1 protein due to the increased level of free transferrin is typical of
iron deficiency anemia Iron-deficiency anemia is anemia caused by a lack of iron. Anemia is defined as a decrease in the number of red blood cells or the amount of hemoglobin in the blood. When onset is slow, symptoms are often vague such as feeling tired, weak, s ...
,
pregnancy Pregnancy is the time during which one or more offspring develops (gestation, gestates) inside a woman, woman's uterus (womb). A multiple birth, multiple pregnancy involves more than one offspring, such as with twins. Pregnancy usually occur ...
, and
oestrogen Estrogen or oestrogen is a category of sex hormone responsible for the development and regulation of the female reproductive system and secondary sex characteristics. There are three major endogenous estrogens that have estrogenic hormonal acti ...
therapy. Increased beta-1 protein due to LDL elevation occurs in
hypercholesterolemia Hypercholesterolemia, also called high cholesterol, is the presence of high levels of cholesterol in the blood. It is a form of hyperlipidemia (high levels of lipids in the blood), hyperlipoproteinemia (high levels of lipoproteins in the blood), ...
. 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 C-reactive protein (CRP) is an annular (ring-shaped) pentameric protein found in blood plasma, whose circulating concentrations rise in response to inflammation. It is an acute-phase protein of hepatic origin that increases following interleukin- ...
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 A skin condition, also known as cutaneous condition, is any medical condition that affects the integumentary system—the organ system that encloses the body and includes skin, nails, and related muscle and glands. The major function of this ...
, or
rheumatoid arthritis Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrist and hands are invol ...
(increased IgA).
Fibrinogen Fibrinogen (factor I) is a glycoprotein complex, produced in the liver, that circulates in the blood of all vertebrates. During tissue and vascular injury, it is converted enzymatically by thrombin to fibrin and then to a fibrin-based blood cl ...
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 An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
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 Hypergammaglobulinemia is a medical condition with elevated levels of gamma globulin. It is a type of immunoproliferative disorder. Types Hypergammaglobulinemia is a condition that is characterized by the increased levels of a certain immunogl ...
), 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 X-linked agammaglobulinemia (XLA) is a rare genetic disorder discovered in 1952 that affects the body's ability to fight infection. As the form of agammaglobulinemia that is X-linked, it is much more common in males. In people with XLA, the whit ...
. 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 An infection is the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmissible disease or communicable dis ...
, chronic liver disease, rheumatoid arthritis, systemic
lupus erythematosus Lupus erythematosus is a collection of autoimmune diseases in which the human immune system becomes hyperactive and attacks healthy tissues. Symptoms of these diseases can affect many different body systems, including joints, skin, kidneys, blo ...
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 Immunofixation permits the detection and typing of monoclonal antibodies or immunoglobulins in Blood serum, serum or urine. It is of great importance for the diagnosis and monitoring of certain blood related diseases such as myeloma. Principle T ...
, 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 Multiple myeloma (MM), also known as plasma cell myeloma and simply myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibodies. Often, no symptoms are noticed initially. As it progresses, bone pain, anemi ...
being the most common cause of IgA and IgG spikes. chronic lymphatic leukaemia and lymphosarcoma are not uncommon and usually give rise to
IgM Immunoglobulin M (IgM) is one of several isotypes of antibody (also known as immunoglobulin) that are produced by vertebrates. IgM is the largest antibody, and it is the first antibody to appear in the response to initial exposure to an antig ...
paraproteins. Note that up to 8% of healthy geriatric patients may have a monoclonal spike. Waldenström's macroglobulinaemia (IgM),
monoclonal gammopathy of undetermined significance Monoclonal gammopathy of undetermined significance (MGUS) is a plasma cell dyscrasia in which plasma cells or other types of antibody-producing cells secrete a myeloma protein, i.e. an abnormal antibody, into the blood; this abnormal protein is ...
(MGUS), amyloidosis, plasma cell leukemia and solitary plasmacytomas also produce an M-spike. Oligoclonal gammopathy is indicated by one or more discrete clones.
Lysozyme Lysozyme (EC 3.2.1.17, muramidase, ''N''-acetylmuramide glycanhydrolase; systematic name peptidoglycan ''N''-acetylmuramoylhydrolase) is an antimicrobial enzyme produced by animals that forms part of the innate immune system. It is a glycoside ...
may be seen as a band cathodal to gamma in myelomonocytic
leukaemia Leukemia ( also spelled leukaemia and pronounced ) is a group of blood cancers that usually begin in the bone marrow and result in high numbers of abnormal blood cells. These blood cells are not fully developed and are called ''blasts'' or ...
in which it is released from
tumour A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...
cells.


References


External links

* Protein electrophoresis a
Lab Tests Online
{{DEFAULTSORT:Serum Protein Electrophoresis Serology Electrophoresis