Electrophoresis & Immunofixation Ramnik Sood, Najat Rashid
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Obstetric Vasculopathies
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Serum Protein Electrophoresis: IntroductionCHAPTER 1

 
INTRODUCTION
Protein electrophoresis is a method for analyzing the proteins in a fluid or an extract. The electrophoresis may be performed in a small volume of sample by a number of alternative ways with or without any supporting media, such as SDS polyacrylamide gel electrophoresis (in short gel electrophoresis, PAGE, or SDS-electrophoresis, free flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, counter electrophoresis, and capillary electrophoresis).
Serum protein electrophoresis is a laboratory examination that is commonly 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 result of this examination can be confusing or difficult to interpret.
Electrophoresis refers to the migration of charged solutes or particles in a liquid medium under the influence of an electrical field. The first electrophoretic method used in the study of proteins was the free solution or moving boundary method devised by Tiselius in 1937. This technique is still used in research for measurement of electrophoretic mobility and for the study of protein-protein interaction but is not used in clinical laboratories for routine work. In this technique, a complex apparatus is needed, the technique is difficult, and sample of approximately 0.5 mL is required.
The term, zone electrophoresis refers to the migration of charged macromolecules in a porous supporting medium, such as cellulose paper, cellulose acetate sheet, or agarose gel film. Zone electrophoresis generates an electrophoretogram, a display of protein zones, each one sharply separated from neighboring zones on the electrophoretic support material. 2Solutes analyzed by this technique in clinical chemistry are mainly macromolecular in size and colloidal in nature. They include proteins in serum, cerebrospinal fluid and other biological fluids, as well as in erythrocytes and tissues.
Chemical species carrying an electric charge by virtue of ionization will move either to the cathode or to the anode in an electrophoresis system, depending on the kind of charge on the molecules. In a solution more acidie than the isoelectric point (pI) of the solute, an ampholyte (a solute molecule that can be either positively or negatively charged, also called a zwitterion) takes on a positive charge by binding protons and migrates toward the cathode (i.e. the negatively charged electrode). Conversely, the ampholyte that has lost protons is in the anionic form and migrates toward the anode (positive electrode) where the protein R is shown with terminal amino (–NH2) and carboxyl (–COOH) groups. Since proteins contain many ionizable groups, this equation should be seen as a simplification. The rate of migration is dependent on such factors as (i) net electrical charge of the molecule, (ii) size and shape of the molecule, (iii) strength of the electrical field, (iv) properties of the supporting medium, and (v) temperature of operation. The equation expressing electrophoretic mobility is a general formula that derives from two formulas, one describing the forward motion toward the electrode of charge opposite to that on the molecule and the other describing the retarding force due to frictional resistance:
Where
μ = electrophoretic mobility, cm/(V)(s)
Q = the net charge on the molecule
K = a constant
r = the ionic radius of the migrating particle
η = the viscosity of the solution in which migration is occurring
Clearly, electrophoretic mobility is directly proportional to the net charge, as the protein migrates under the influence of an applied electric current and inversely proportional to the size of the protein ion and the viscosity of the medium (electrophoretic buffer).
This book provides a comprehensive review of serum protein electrophoresis, including a discussion of how the examination is performed, what it measures, and when it is indicated. It provides a simple guide to result interpretation and suggestions on follow-up of abnormal results.
 
COMPONENTS OF SERUM PROTEIN ELECTROPHORESIS
The pattern of serum protein electrophoresis results depends on the fractions of two major types of proteins—albumin and globulin. Albumin, the major protein component of serum, is produced by the liver under normal physiologic condition. Globulins comprise a much smaller fraction of the total serum protein content. The subsets of these proteins and their 3relative quantity are the primary focus of the interpretation of serum protein electrophoresis. Albumin, the largest peak, lies closest to the positive electrode. The next five components (globulins) are labeled alpha 1 (α1), alpha 2 (α2), beta 1 (β1), beta 2 (β2), and gamma (γ). The peaks for these components lie toward the negative electrode, with the γ peak being closest to that electrode (Figs 1 and 2).
 
Prealbumin
Prealbumin is the fraction migrating faster than albumin toward the anode. It is same as transthyretin (TTR). Mutations in transthyretin are associated with hereditary amyloidosis. True prealbumin is generally below the limit of detection in serum protein electrophoresis but may be detected in electrophoresis of concentrated cerebrospinal fluid (CSF).
 
Albumin
Albumin represents the largest protein component of human serum that is water soluble, is moderately soluble in concentrated salt solutions, and experiences heat denaturation.
Fig. 1: Protein electrophoresis components
Fig. 2: Typical normal pattern for serum protein electrophoresis
4
Albumins are commonly found in blood plasma and are unique from other blood proteins, as they are not glycosylated. Substances containing albumin, such as egg white, are called albuminoids.
Albumin is the main protein of plasma. It binds water, cations (such as Ca2+, Na+ and K+), fatty acids, hormones, bilirubin, thyroxine (T4) and drugs (including barbiturates). Its main function is to regulate the colloidal osmotic pressure of blood. a- fetoprotein (α-fetoglobulin) is a fetal plasma protein that binds various cations, fatty acids, and bilirubin. Vitamin D-binding protein binds to vitamin D and its metabolites, as well as to fatty acids. The biological role of afamin (α-albumin) has not yet been characterized.
Albumin comprises three homologous domains that assemble to form a heart-shaped molecule. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and IIIA that exhibit similar chemistry. Structurally, the serum albumins are similar, each domain containing five or six internal disulfide bonds as shown in Figure 3.
Serum albumins are important in regulating blood volume by maintaining the oncotic pressure (also known as colloid osmotic pressure) of the blood compartment. They also serve as carriers for molecules of low water solubility this way isolating their hydrophobic nature, including lipid soluble hormones, bile salts, unconjugated bilirubin, free fatty acids (apoprotein), calcium, ions (transferrin) and some drugs like warfarin, phenobutazone, clofibrate and phenytoin. For this reason, it is sometimes referred as a molecular “taxi”. Competition between drugs for albumin binding sites may cause drug interaction by increasing the free fraction of one of the drugs.
Fig. 3: Movement of proteins electrophoretically
(Abbreviations: C: cysteine; x: any amino acid)
5
Normal range of human serum albumin in adults is 3.5–5 g/dL. For children less than three years of age, the normal range is broader, 2.9–5.5 g/dL.
A decreased albumin level may be indicative of liver failure or diseases such as cirrhosis or chronic hepatitis. Hypoalbuminemia can also present as part of the nephrotic syndrome, in which protein is lost in the urine, because of kidney damage. Low albumin levels can be an indicator of chronic malnutrition or protein losing enteropathy, hormone therapy, and pregnancy. Burns also may result in a low albumin level.
Hypoalbuminemia may cause generalized edema (swelling) via a decrease in oncotic pressure.
Levels below 3.5 g/dL are generally considered low. At the time of hospital admission, 20% of patients have hypoalbuminemia. Serum albumin level is an important prognostic indicator. Among hospitalized patients, lower serum albumin levels correlate with an increased risk of morbidity and mortality.
Absence of albumin, known as analbuminemia, is a benign condition. It is a genetically inherited metabolic defect characterized by an impaired synthesis of serum albumin.
Levels of albumin are increased (hyperalbuminemia) in patients with a relative reduction in serum water (e.g. dehydration). In some cases of retinol (v itamin A) deficiency of the albumin level can be elevated to high normal values (e.g. 4.9 g/dL). This is because retinol causes cells to swell with water (this is also the reason because excess vitamin A is toxic).
Usually a single band is seen, individuals may produce bisalbuminemia (alloalbminemia), a condition in which two types of albumin exist in an individual. It is considered as an autosomal dominant condition of having serum albumin of a variant type, which differs in mobility on electrophoresis and affects people who are heterozygous or homozygous for one of the alleles for variant albumin types.
 
Albumin Alpha 1 Interzone
Even staining in this zone is due to alpha 1 (α1) lipoprotein [high density lipoprotein (HDL)]. Decrease occurs in severe inflammation, acute hepatitis and cirrhosis. Also, nephrotic syndrome can lead to a 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 alpha fetoprotein (AFP) that may occur in hepatocellular carcinoma may result in a sharp band between the albumin and the α1 zone.
 
Alpha Zone
Moving toward the negative portion of the gel (i.e. the negative electrode), the next peaks involve the α1 and α2 components. The α1 protein fraction is comprised of α1 antitrypsin, thyroid binding globulin and transcortin. Malignancy and acute inflammation (resulting from acute phase reactants) can increase the α1 protein band. A decreased α1 protein band may 6occur because of α1 antitrypsin deficiency or decreased production of the globulin as a result of liver disease. Ceruloplasmin, α2 macroglobulin, and haptoglobin (Hp) contribute to the α2 protein band, and the α2 component is increased as an acute-phase reactant.
 
Alpha 1 (α1) Zone
Orosomucoid and antitrypsin migrate together but orosomucoid stains poorly so α1 antitrypsin constitutes most of the α1 band. α1 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 α1 antitrypsin deficiency. This eventually leads to emphsysema from unregulated lung elastase breakdown by neutrophils in the lung tissue. The α1 fraction does not disappear in α1 antitrypsin deficiency; however, because other proteins, including α lipoprotein and α1 acid glycoprotein, also migrate there. As a positive acute phase reactant, α1 antitrypsin is increased in acute inflammation.
 
Alpha 1 (α1)Antitrypsin
α1 antitrypsin (α1AT) is a protease inhibitor belonging to the serpin superfamily. This was originally named “antitrypsin” because of its ability to covalently bind and irreversibly inactivate the enzyme trypsin in vitro. Trypsin, a type of peptidase, is a digestive enzyme active in the duodenum and elsewhere. The term α1 refers to the protein's behavior on protein electrophoresis. On electrophoresis, the protein component of the blood is separated by electric current. There are several clusters, the first being albumin, the second alpha (α), the third beta (β), and the fourth gamma (γ) (immunoglobulins). The nonalbumin proteins are referred to as globulins. The α region can be further divided into two subregions, termed α1 and α2. α 1 antitrypsin is the main protein of the α globulin 1 region. Another name used is α 1 proteinase inhibitor (α1 PI).
It is generally known as serum trypsin inhibitor. Alpha 1 antitrypsin is also referred to as α1 proteinase inhibitor because it inhibits a wide variety of proteases. It protects tissues from enzymes of inflammatory cells, especially neutrophil elastase and has a reference range in blood of 1.5–3.5 g/L (in US the reference range is generally expressed as mg/dL or micromoles), but the concentration can rise manyfold upon acute inflammation. In its absence, neutrophil elastase is free to break down elastin, which contributes to the elasticity of the lungs, resulting in respiratory complications such as emphysema, or chronic obstructive pulmonary disease (COPD) in adults and cirrhosis in adults or children.
α 1 antitrypsin is a 52 k D serpin, and in medicine, it is considered the most prominent serpin. The terms α1 antitrypsin and protease inhibitor (PI) are often used interchangeably. Most serpins inactivate enzymes by binding to them covalently, requiring very high levels to perform their function. In the acute phase reaction, a further elevation is required to “limit” the damage 7caused by activated neutrophil granulocytes and their enzyme elastase that breaks down the connective tissue fiber elastin. Like all serine protease inhibitors, α1AT has a characteristic secondary structure of β sheets and α helices. Mutations in these areas can lead to nonfunctional proteins that can polymerize and accumulate in the liver (infantile hepatic cirrhosis).
Disorders of this protein include α1 antitrypsin deficiency, a hereditary disorder in which a deficiency of α1 antitrypsin leads to a chronic uninhibited tissular breakdown. This causes the degradation especially of lung tissue and eventually leads to characteristic manifestations of pulmonary emphysema. Evidence has shown that cigarette smoke can lead to oxidation of methionine 358 of α1 antitrypsin, a residue essential for binding elastase. This is thought to be one of the primary mechanisms by which cigarette smoking (or second hand smoke) can lead to emphysema. Because α1AT is expressed in the liver, certain mutations in the gene encoding the protein can cause misfolding and impaired secretion, which can lead to liver cirrhosis.
 
Alpha 1 (α1) and Alpha 2 (α2) Interzone
Two faint bands may be seen representing α1 antichymotrypsin and vitamin D binding protein. These bands fuse and intensify in early inflammation, because of an increase in α1 antichymotrypsin, an acute phase protein.
 
Alpha 2 Zone
This zone consists principally of α2 macroglobulin and Hp. There are typically low levels in hemolytic anemia [Hp is a suicide molecule, which binds with free hemoglobin (Hb) 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 α2 zone during inflammation. A normal α2 and an elevated α1 zone is a typical pattern in hepatic metastasis and cirrhosis.
 
Haptoglobin
Haptoglobin/hemoglobin complexes migrate more cathodally than Hp as seen in the α2-β interzone. This is typically seen as a broadening of the α2 zone. Haptoglobin is a protein that in humans is encoded by the HP gene. In blood plasma, Hp binds free Hb released from erythrocytes with high affinity and thereby inhibits its oxidative activity. The Hp Hb complex will then be removed by the reticuloendothelial system (mostly the spleen). In clinical settings, the haptoglobulin assay is used to screen for and monitor intravascular hemolytic anemia. In intravascular hemolysis free Hb will be released into circulation and hence Hp will bind the Hb. This causes a decline in Hp levels. Conversely, in extravascular hemolysis, the reticuloendothelial system, especially splenic monocytes, phagocytose the erythrocytes and Hb is not released into circulation; therefore, serum Hp levels are normal. Haptoglobin is produced mostly by hepatocytes but also by other tissues, e.g. skin, lung and kidney. In addition, the haptoglobin gene is expressed in murine and human adipose tissue and had been shown to be expressed in 8adipose tissue of cattle as well. Haptoglobin, in its simplest form, consists of two α and two β chains, connected by disulfide bridges. The chains originate from a common precursor protein, which is proteolytically cleaved during protein synthesis. Haptoglobin exists in two allelic forms in the human population, so called Hp1 and Hp2, the latter have arisen because of the partial duplication of Hp1 gene. Three genotypes of Hp, therefore, are found in humans: Hp1-1, Hp2-1 and Hp2-2. Haptoglobin of different genotypes has been shown to bind Hb with different affinities, with Hp2-2 being the weakest binder.
Mutations in this gene and/or its regulatory regions cause haptoglobinemia or hypohaptoglobinemia. This gene has also been linked to diabetic nephropathy, the incidence of coronary artery disease in type 1 diabetes mellitus, Crohn's disease, inflammatory disease behavior, primary sclerosing cholangitis, susceptibility to idiopathic Parkinson's disease and a reduced incidence of Plasmodium falciparum malaria.
Since the reticuloendothelial system will remove the haptoglobin-hemoglobin complex from the body, haptoglobin levels will be decreased in hemolytic anemia. In the process of binding Hb, Hp sequesters the iron within Hb, preventing iron utilizing bacteria in benefiting from hemolysis. It is theorized that, because of this, haptoglobin has evolved into an acute phase protein. Haptoglobin has a protective influence on the hemolytic kidney. Some studies associate certain Hp phenotypes with the risk of developing schizophrenia.
Haptoglobin is ordered whenever a patient exhibits symptoms of anemia, such as pallor, fatigue, or shortness of breath, along with physical signs of hemolysis, such as jaundice or dark-colored urine. The test is also commonly ordered as a hemolytic anemia battery, that also includes a reticulocyte count and a peripheral blood smear. It can also be ordered along with a direct antiglobulin test when a patient is suspected of having a transfusion reaction or symptoms of autoimmune hemolytic anemia. Also, it may be ordered in conjunction with a bilirubin.
A decrease in Hp can support a diagnosis of hemolytic anemia, especially when correlated with a decreased red blood cell count, haemoglobin and hematocrit, and also an increased reticulocyte count.
If the reticulocyte count is increased, but the Hp level is normal, this may indicate that cellular destruction is occurring in the spleen and liver, which may indicate a drug-induced hemolysis or a red-cell dysplasia. The spleen and liver recognize an error in the red cells (either drug coating the red cell membrane or a dysfunctional red cell membrane) and destroy the cell. This type of destruction does not release Hb into the peripheral blood, so the Hp cannot bind to it. Thus, the Hp will stay normal, if the hemolysis is not severe. In severe extravascular hemolysis, Hp levels can also be low, when large amount of hemoglobin in the reticuloendothelial system leads to transfer of free Hb into plasma. If there are symptoms of anemia, and both the reticulocyte count and the Hp level are normal, the anemia is most likely not due to hemolysis, but instead some other error in cellular production, such as aplastic anemia. Haptoglobin levels that are decreased but do not 9accompany signs of anemia may indicate liver damage, as the liver is not producing enough Hp to begin with. As Hp is indeed an acute phase protein, any inflammatory process (infection, extreme stress, burns, major crush injury, allergy, etc.) may increase the levels of plasma Hp.
 
Alpha 2 (α2) Macroglobulin
α2 macroglobulin is a large plasma protein found in the blood. It is produced by the liver and is a major component of the α2 band in protein electrophoresis. It is the largest major nonimmunoglobulin protein in plasma. The α2 macroglobulin molecule is synthesized mainly in liver, but also locally by macrophages, fibroblasts, and adrenocortical cells.
α2 macroglobulin acts as an antiprotease and is able to inactivate an enormous variety of proteinases. It functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein. It functions as an inhibitor of coagulation by inhibiting thrombin. α2 macroglobulin may act as a carrier protein because it also binds to numerous growth factors and cytokines, such as platelet-derived growth factor, basic fibroblast growth factor, tumor growth factor β (TGF β), insulin, and Interleukin 1 β (IL 1β).
No specific deficiency with associated disease has been recognized, and no disease state is attributed to low concentrations of α2 macroglobulin. The concentration of α2 macroglobulin raises 10-fold or more in the nephrotic syndrome when other lower molecular weight proteins are lost in the urine. The loss of α2 macroglobulin into urine is prevented by its large size. The net result is that α2 macroglobulin reaches serum levels equal to or greater than those of albumin in the nephrotic syndrome, which has the effect of maintaining oncotic pressure.
Human α2 macroglobulin is composed of four identical subunits bound together by S-S bonds. In addition to tetrameric forms of α2 macroglobulin, dimeric and more recently monomeric aM protease inhibitors have been identified. Each monomer of human α2 macroglobulin is composed of many functional domains, including macroglobulin domains, a thiol ester containing domain and a receptor binding domain.
α 2 macroglobulin is able to inactivate an enormous variety of proteinases (including serine, cysteine, aspartic and metalloproteinases). It functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein (Fig. 4). It functions as an inhibitor of coagulation by inhibiting thrombin.
α 2 macroglobulin may be elevated in children and the elderly. This is seen as a sharp front to the α2 band. α2 macroglobin is markedly raised (10-fold increase or greater) in association with glomerular protein loss, as in nephrotic syndrome. Due to its large size, α2 macroglobulin cannot pass through glomeruli, while other lower molecular weight proteins are lost. Enhanced synthesis of α2 macroglobulin accounts for its absolute increase in nephrotic syndrome wherein the kidneys start to leak out some of the smaller blood proteins. Because of its size, α2 macroglobulin is retained in the bloodstream. Increased production of all proteins means α2 macroglobulin concentration increases. This increase has little adverse effect on the health, 10but is used as a diagnostic clue.
Fig. 4: Fibrinolysis (simplified). Blue arrows denote stimulation and red arrows denote inhibition
A common variant (29.5%) (polymorphism) of α2 macroglobulin leads to increased risk of Alzheimer's disease and mildly elevated early in the course of diabetic nephropathy.
 
Ceruloplasmin
Ceruloplasmin is the major copper -carrying protein in the blood and in addition plays a role in iron metabolism. It was first described in 1948. The molecular weight of human ceruloplasmin is reported to be 151 kD. Another protein, hephaestin, is noted for its homology to ceruloplasmin and also participates in iron and probably copper metabolism. Ceruloplasmin is an enzyme (EC 1.16.3.1) synthesized in the liver containing six atoms of copper in its structure. Ceruloplasmin carries about 70% of the total copper in human plasma, while albumin carries about 15%. The rest is accounted for by macroglobulins. Albumin may be confused at times to have a greater importance as a copper carrier, because it binds copper less tightly than ceruloplasmin. Ceruloplasmin exhibits a copper-dependent oxidase activity, which is associated with possible oxidation of Fe2+ (ferrous iron) into Fe3+ (ferric iron), therefore assisting in its transport in the plasma in association with transferrin that can carry iron only in the ferric state.
Copper availability does not affect the translation of the nascent protein. However, the apoenzyme without copper is unstable. Apoceruloplasmin is largely degraded intracellularly in the hepatocyte, and the small amount that is released has a short circulation half life of 5 hours as compared to the 5.5 days for the holo-ceruloplasmin.11
Mutations in the ceruloplasmin gene (CP), which are very rare, can lead to the genetic disease aceruloplasminemia, characterized by hyperferritinemia with iron overload. In the brain, this iron overload may lead to characteristic neurologic signs and symptoms, such as cerebellar ataxia, progressive dementia, and extrapyramidal signs. Excess iron may also deposit in the liver, pancreas and retina, leading to cirrhosis, endocrine abnormalities and loss of vision, respectively.
Like any other plasma protein, levels drop in patients with hepatic disease because of reduced synthesizing capabilities and the low levels may indicates Wilson disease, Menkes disease, overdose of v itamin C, c opper deficiency, a ceruloplasminemia. On the other hand, greater levels than normal may indicate pregnancy, oral contraceptive pill, lymphoma, rheumatoid arthritis, angina, Alzheimer's disease, schizophrenia, obsessive-compulsive disorder, and acute and chronic inflammation.
 
Alpha 2-(α2) 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. β lipoprotein forms an irregular crenated band in this zone. High levels are seen in type II hypercholesterolemia, hypertriglyceridemia, and in the nephrotic syndrome.
 
Beta Zone
The β fraction has two peaks labeled β1 and β2. b1 is composed mostly of transferrin, and β2 contains β lipoprotein. Immunoglobulin A (IgA), IgM, and sometimes IgG, along with complement proteins, also can be identified in the β fraction. Transferrin and β lipoprotein [low-density lipoprotein (LDL )] comprises β1. Increased β1 protein due to the increased level of free transferrin is typical of iron deficiency anemia, pregnancy, and estrogen therapy. Increased β1 protein due to LDL elevation occurs in hypercholesterolemia.
β2 comprises c omplement protein 3 (C3). 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 plasma. Fibrinogen, a β2 protein, is found in normal plasma but also absent from normal serum. Occasionally, blood drawn from heparinized patients, does not fully clot, resulting in a visible fibrinogen band between the β and γ globulins.
 
Complement Component 3
Complement component 3 is a protein of the immune system. It plays a central role in the complement system and contributes to innate immunity. In humans, it is encoded on chromosome 19 by a gene called C3. C3 plays a central role in the activation of complement system. Its activation is required for both classical and alternative complement activation pathways. People with C3 deficiency are susceptible to bacterial infection.12
One form of C3 convertase, also known as C4b2a, is formed by a heterodimer of activated forms of C4 and C2. It catalyzes the proteolytic cleavage of C3 into C3a and C3b, generated during activation through the classical pathway as well as the mannan binding lectin pathway. C3a is an anaphylotoxin and the precursor of some cytokines, such as ASP and C3b, serves as an opsonizing agent. Factor I can cleave C3b into C3c and C3d in which the latter plays a role in enhancing B cell responses. In the alternative complement pathway, C3 is cleaved by C3bBb, another form of C3 convertase composed of activated forms of C3 (C3b) and factor B (Bb). Once C3 is activated to C3b, it exposes a reactive thioester that allows the peptide to covalently attach to any surface, which can provide a nucleophile, such as a primary amine or a hydroxyl group. Activated C3 can then interact with factor B. Factor B is then activated by factor D to form Bb. The resultant complex, C3bBb, is called the alternative pathway (AP) C3 convertase.
C3bBb is deactivated in steps. First, the proteolytic component of the convertase, Bb, is removed by complement regulatory proteins having decay accelerating factor (DAF) activity. Next, C3b is broken down progressively to first iC3b, then C3c + C3dg, and then finally C3d. Factor I is the protease that performs these cuts but it requires the help of another protein to supply what is termed cofactor activity. Several crystallographic structures of C3 have been determined and reveal that this protein contains 13 domains. Levels of C3 in the blood may be measured to support or refute a particular medical diagnosis. For example, low C3 levels are associated with some types of kidney disease, such as postinfectious glomerulonephritis and shunt nephritis.
 
Gamma (γ) Zone
Much of the clinical interest is focused on the γ 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 β and γ components. The immunoglobulins (IgA, IgM, IgG, IgE and IgD) are the only proteins present in the normal γ region, but note that immunoglobulins may be found in the α and β zones. If the γ zone shows an increase (or spike), the first step in interpreting the graph is to establish if the region is narrow or wide. If it is elevated it could be elevated in a single narrow “spike like” manner indicating monoclonal gammopathy or a broad «swell like» manner (wide) indicating polyclonal gammopathy.
 
Monoclonal Gammopathy
A narrow spike indicates a monoclonal gammopathy, also known as an “M spike”. Typically, a monoclonal gammopathy is malignant or clonal in origin, myeloma being the most common cause of IgA and IgG spikes. Chronic lymphatic leukemia 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 macroglobulinemia (WM) IgM, monoclonal gammopathy of undetermined significance (MGUS), amyloidosis, plasma cell leukemia and solitary plasmacytomas also produce an M spike.13
 
Polyclonal Gammopathy
A “swell like” elevation in the γ zone indicates a polyclonal gammopathy, and this typically points to a nonmalignant condition (although is not exclusive to nonmalignant conditions). The most common causes of polyclonal hypergammaglobulinemia detected by electrophoresis are severe infection, chronic liver disease, rheumatoid arthritis, systemic lupus erythematosus and other connective tissue diseases. Lysozyme may be seen as a band cathodal to the slowest γ in myelomonocytic leukemia in which it is released from tumor cells. Fibrinogen from plasma samples will be seen in the fast γ region.
 
Hypogammaglobulinemia
This is easily identifiable as a “slump” or decrease in the γ 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 pallor in the γ zone.
 
Immunoglobulin A
Immunoglobulin A (IgA) antibodies are found in areas of the body, such as the nose, breathing passages, digestive tract, ears, eyes and vagina. IgA antibodies protect body surfaces that are exposed to outside foreign substances. This type of antibody is also found in saliva, tears, and blood. About 10–15% of the antibodies present in the body are IgA antibodies. A small number of people do not make IgA antibodies. It plays a critical role in mucosal immunity, more IgA is produced in mucosal linings than all other types of antibodies combined: between 3 g and 5 g are secreted into the intestinal lumen each day. This accumulates to 75% of the total immunoglobulin produced in the entire body. IgA has two subclasses (IgA1 and IgA2) and can exist in a dimeric form called secretory IgA (sIgA).
In its secretory form (Fig. 5), IgA is the main immunoglobulin found in mucous secretions, including tears, saliva, colostrum, and secretions from the genitourinary tract, gastrointestinal tract, prostate, and respiratory epithelium. It is also found in small amounts in blood. The secretory component of sIgA protects the immunoglobulin from being degraded by proteolytic enzymes; 14thus, sIgA can survive in the harsh gastrointestinal tract environment and provide protection against microbes, which multiply in body secretions.
Fig. 5: Immunglobulin A structure as a dimer
IgA is a poor activator of the complement system and opsonies only weakly. Its heavy chains are of the type α. This immunoglobulin has the most anodal mobility and migrates in the region between the β and γ zones also causing a β/γ fusion in patients with cirrhosis, respiratory infection, skin disease or rheumatoid arthritis (increased IgA).
Immunoglobulin A exists in two isotypes : IgA1 and IgA2. While IgA1 predominates in serum (~80%), IgA2 percentages are higher in secretions than in serum (~35% in secretions). The ratio of IgA1 and IgA2 secreting cells varies in the different lymphoid tissues of the human body:
  • IgA1 is the predominant IgA subclass found in serum. Most lymphoid tissues have a predominance of IgA producing cells
  • In IgA2, the heavy and light chains are not linked with disulfide, but with noncovalent bonds. In secretory lymphoid tissues [e.g. gut associated lymphoid tissue (GALT)], the share of IgA2 production is larger than in the nonsecretory lymphoid organs (e.g. spleen, peripheral lymph nodes).
Both IgA1 and IgA2 have been found in external secretions like colostrum, maternal milk, tears and saliva, where IgA2 is more prominent than in the blood. Polysaccharide antigens tend to induce more IgA2 than protein antigens.
It is also possible to distinguish forms of IgA based upon their location serum IgA vs secretory IgA.
In secretory IgA, the form found in secretions, polymers of two to four IgA monomers are linked by two additional chains; as such sIgA holds a molecular weight of 385,000. One of these is the joining chain (J chain), which is a polypeptide of molecular mass 15 kD, rich with cysteine and structurally completely different from other immunoglobulin chains. This chain is formed in the IgA secreting cells.
The oligomeric forms of IgA in the external (mucosal) secretions also contain a polypeptide of a much larger molecular mass (70 kD) called the secretory component, which is produced by epithelial cells. This molecule originates from the poly Ig receptor (130 kD) that is responsible for the uptake and transcellular transport of oligomeric (but not monomeric) IgA across the epithelial cells and into secretions, such as tears, saliva, sweat, and gut fluid.
The high prevalence of IgA in mucosal areas is a result of cooperation between plasma cells that produce polymeric IgA (pIgA) and mucosal epithelial cells that express an immunoglobulin receptor called the polymeric Ig receptor (pIgR). Polymeric IgA is released from the nearby activated plasma cells and binds to pIgR. This results in transportation of IgA across mucosal epithelial cells and its cleavage from pIgR for release into external secretions.
In the blood, IgA interacts with an Fc receptor called FcαRI (or CD89), which is expressed on immune effector cells, to initiate inflammatory reactions. Ligation of FcαRI by IgA containing immune complexes cause 15antibody dependent cell-mediated cytotoxicity (ADCC), degranulation of eosinophils and basophils, phagocytosis by monocytes, macrophages and neutrophils, and triggering of respiratory burst activity by polymorph nuclear leukocytes.
Polymeric IgA (mainly the secretory dimer) is produced by plasma cells in the lamina propria, adjacent to mucosal surfaces. It binds to the polymeric immunoglobulin receptor on the basolateral surface of epithelial cells and is taken up into the cell via endocytosis. The receptor IgA complex passes through the cellular compartments before being secreted on the luminal surface of the epithelial cells, still attached to the receptor. Proteolysis of the receptor occurs, and the dimeric IgA molecule, along with a portion of the receptor known as the secretory component, are free to diffuse throughout the lumen. In the gut, it can bind to the mucous layer on top of the epithelial cells to form a barrier capable of neutralizing threats before they reach the cells. Decreased or absent IgA, termed selective IgA deficiency, can be a clinically significant immunodeficiency.
Neisseria gonorrhoeae (that causes gonorrhea), Streptococcus pneumoniae, and Haemophilus influenzae type B all release a protease which, destroys IgA.
Immunoglobulin A nephropathy is caused by IgA deposits in the kidneys. It is not yet known why IgA deposits occur in this chronic disease. Some theories suggest an abnormality of the immune system results in these deposits.
Celiac disease involves IgA pathology due to the presence of IgA antiendomysial antibodies.
 
Clinical Significance
The IgA fixes the complement by the alternative path: polyclonal increases of IgA can be present in chronic inflammatory panels of the respiratory tract and the gastroenteric tract, including the liver. About one-fourth of the patients with IgA deficit have anti-IgA antibodies and are at risk for serious anaphylactic reactions in case of blood/plasma transfusions.
Increased IgA levels—chronic hepatopathy, cirrhosis, chronic respiratory infections, neoplasia of the final section of the intestine, gastroenteric apparatus diseases (Crohn's disease, ulcerative colitis, etc.), rheumatoid arthritis, ankylosing spondylitis, neuropathies, Wiskott Aldrich syndrome; IgA myeloma, MGUS (monoclonal).
Decreased IgA levels—infancy, selective deficiency of IgA (approximately 1/700), protide dispersion syndrome, macroglobulinemia, or non-IgA myeloma.
 
Immunoglobulin G
Immunoglobulin G (IgG) antibodies are found in all body fluids. They are the smallest but most common antibody (75–80%) of all the antibodies in the body. IgG antibodies are very important in fighting bacterial and viral infections. IgG antibodies are the only antibody that can cross the placenta in a pregnant woman to help protect her baby (fetus).16
Fig. 6: Immunoglobulin G structure
IgG is composed of four peptide chains two heavy chains γ and two light chains (Fig. 6). Each IgG has two antigen -binding sites. Other immunoglobulins may be described in terms of polymers with the IgG structure considered the monomer.
Ig G constitutes 75% of serum immunoglobulins in humans. IgG molecules are synthesized and secreted by plasma B cells. IgG antibodies are involved predominantly in the secondary immune response. The presence of specific IgG, in general, corresponds to maturation of the antibody response.
 
Human Immunoglobulin G Subclasses
Immunoglobulin G is the only isotype that can pass through the human placenta, thereby, providing protection to the fetus in utero. Along with IgA secreted in the breast milk, residual IgG absorbed through the placenta provides the neonate with humoral immunity before its own immune system develops. Colostrum contains a high percentage of IgG, especially bovine colostrum.
Immunoglobulin G can bind to many kinds of pathogens, (for example, viruses, bacteria and fungi) and protects the body against them by agglutination and immobilization, complement activation (classical pathway), opsonization for phagocytosis, and neutralization of their toxins. It also plays an important role in ADCC and intracellular antibody mediated proteolysis, in which it binds to TRIM21 (the receptor with greatest affinity to IgG in humans) in order to direct marked virions to the proteasome in the cytosol, IgG is also associated with type II and type III hypersensitivity.
Immunoglobulin G antibodies are large molecules of about 150 k D composed of four peptide chains. It contains two identical class γ heavy chains of about 50 kD and two identical light chains of about 25 kD, thus a tetrameric quaternary structure. The two heavy chains are linked to each other and to a light chain each by disulfide bonds. The resulting tetramer has two identical halves, which together form a Y like shape. Each end of the fork contains an identical antigen binding site. The Fc regions of IgGs bear a highly conserved N glycosylation site. The N glycans attached to this site are predominantly core fucosylated diantennary structures of the complex type. In addition, small amounts of these N glycans also bear bisecting GlcNAc and α 2, 6 linked sialic acid residues.17
Immunoglobulin G has a low frequency wave number of 28 cm−1 in the Raman spectra. This emission has been assigned to the breathing motion in the β barrel of nine β strands in its VH domain. The dynamic mechanism of the “chelate effect” and “trigger effect” of IgG has been analyzed from the angle of low frequency resonance among the 12 β barrels of an IgG molecule.
There are four IgG subclasses (IgG1, IgG2, IgG3, and IgG4) in humans, named in order of their abundance in serum (IgG1 being the most abundant).
Immunoglobulin G affinity to Fc receptors on phagocytic cells is specific to individual species from which the antibody comes as well as the class. The structure of the hinge regions gives each of the four IgG classes its unique biological profile. Even though there is about 95% similarity between their Fc regions, the structure of the hinge regions is relatively different.
In a model of autoantibody mediated anemia using IgG isotype switch variants of an antierythrocytes autoantibody, it was found that IgG2a was superior to IgG1 in activating complement. Moreover, it was found that the IgG2a isotype was able to interact very efficiently with Fc-γ receptors (FcγR). As a result, 20 times higher doses of IgG1 in relationship to IgG2a autoantibodies, were required to induce autoantibody mediated pathology.
 
Clinical Significance
The IgG represent from 75 to 80% of the total immunoglobulins, fix the complement through the classic path (IgG1, IgG2, IgG3) and alternative path (IgG4) and constitute the specific immune response. They are the only immunoglobulin, which passes the placenta. Deficiency of IgG is associated with repeated serious infections.
Increased IgG levels—autoimmune diseases [Lambert-Eaton syndrome (LES), AR, systemic sclerosis, Sjogren syndrome, etc.), chronic hepatopathy, recurrent or chronic infections, sarcoidosis, intrauterine contraceptive devices.
Oligoclonal: lymphoid and nonlymphoidal neoplasia, viral infections, autoimmune diseases
Monoclonal: IgG myeloma, MGUS, lymphomas
Decreased IgG levels—infancy, pregnancy (moderate), hypogammaglobulinemia, agammaglobulinemia, nephrotic syndrome, non-IgG myelomas.
 
Immunoglobulin M
Immunoglobulin M antibodies are the largest antibody. They are found in blood and lymph fluid and are the first type of antibody made in response to an infection. They also cause other immune system cells to destroy foreign substances. IgM antibodies are about 5–10% of all the antibodies in the body.
Immunoglobulin M is produced by B cells ; it is by far the physically largest antibody in the human circulatory system. It is the first antibody to appear in response to initial exposure to antigen. The spleen is the major site of specific IgM production. IgM forms polymers where multiple immunoglobulins are covalently linked together with disulfide bonds, 18mostly as a pentamer but also as a hexamer.
Fig. 7: A pentameric immunoglobulin M
IgM has a molecular mass of approximately 900 k D (in its pentamer form). Because each monomer has two antigen binding sites, a pentameric IgM has 10 binding sites (Fig. 7). Typically, however, IgM cannot bind 10 antigens at the same time because the large size of most antigens hinders binding to nearby sites.
The J chain is found in pentameric IgM but not in the hexameric form, perhaps due to space constraints in the hexameric complex. Pentameric IgM can also be made in the absence of J chain. At present, it is still uncertain what fraction of normal pentamer contains J chain, and to this extent, it is also uncertain whether a J chain containing pentamer contains one or more than one J chain. Because IgM is a large molecule, it cannot diffuse well and is found in the interstitium only in very low quantities. IgM is primarily found in serum ; however, because of the J chain, it is also important as a secretory immunoglobulin. Due to its polymeric nature, IgM possesses high avidity and is particularly effective at complement activation. By itself, IgM is an ineffective opsonin ; however, it contributes greatly to opsonization by activating complement and causing C3b to bind to the antigen. In germline cells, the gene segment encoding the μ constant region of the heavy chain is positioned first among other constant region gene segments. For this reason, IgM is the first immunoglobulin expressed by mature B cells. It is also the first immunoglobulin expressed in the fetus (around 20 weeks) and also phylogenetically the earliest antibody to develop.
Immunoglobulin M antibodies appear early in the course of an infection and usually reappear, to a lesser extent, after further exposure. IgM antibodies do not pass across the human placenta (only isotype IgG).
These two biological properties of IgM make it useful in the diagnosis of infectious diseases. Demonstrating IgM antibodies in a patient's serum indicates recent infection, or in a neonate's serum indicates intrauterine infection (e.g. congenital rubella).
 
Clinical Significance
The IgM fixes the complement through the classic path and constitutes the primary response to the infections. Viral IgM specific antibodies in 19newborns are due to congenital infections (they do not pass through the placenta filter).
Increased IgM levels: Viral infections, parasitosis, chronic hepatopathy, primitive biliary cirrhosis, primary sclerosing cholangitis.
Monoclonal: Waldenstrom macroglobulinemia, malignant lymphoma, reticulosis, cryoglobulins, cryoagglutinins.
Decreased IgM levels: Infancy, immunodeficiency conditions (Wiskott-Aldrich syndrome), non-IgM myeloma.
 
C-reactive Protein
Discovered by Gillett and Francis in 1930, it was initially thought that CRP might be a pathogenic secretion, as it was elevated in people with a variety of illnesses, including cancer, however, discovery of hepatic synthesis demonstrated that it was a native protein, CRP was so named because it was first discovered as a substance in the serum of patients with acute inflammation that reacted with the C (capsular) polysaccharide of P neumococcus.
C-reactive protein is a protein found in the blood, the levels of which rise in response to inflammation (i.e. C-reactive protein is an acute-phase protein). Its physiological role is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system via the C1Q complex.
C-reactive protein is synthesized by the liver in response to factors released by macrophages and fat cells (adipocytes). It is a member of the pentraxin family of proteins, and it is not related to C peptide or protein C. C-reactive protein was the first pattern recognition receptor (PRR) to be identified. The CRP gene is located on the first chromosome (1q21-q23). CRP is a 224 residue protein with a monomer molar mass of 25106 Da. The protein is an annular pentameric disk in shape and a member of the small pentraxins family.
The acute phase response develops in a wide range of acute and chronic inflammatory conditions like bacterial, viral or fungal infections; rheumatic and other inflammatory diseases; malignancy; and tissue injury or necrosis. These conditions cause release of IL 6 and other cytokines, which trigger the synthesis of CRP and fibrinogen by the liver. During the acute phase response, levels of CRP rapidly increase within 2 hours of acute insult, reaching a peak at 48 hours. With resolution of the acute phase response, CRP declines with a relatively short half-life of 18 hours. Measuring CRP level is a screen for infectious and inflammatory diseases. Rapid, marked increases in CRP occur with inflammation, infection, trauma and tissue necrosis, malignancies, and autoimmune disorders. Because there are a large number of disparate conditions, which can increase CRP production, an elevated CRP level does not diagnose a specific disease. An elevated CRP level can provide support for the presence of an inflammatory disease, such as rheumatoid arthritis, polymyalgia rheumatica, or giant cell arteritis.
The physiological role of CRP is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system. CRP binds to phosphocholine on microbes 20and damaged cells and enhances phagocytosis by macrophages. Thus, CRP participates in the clearance of necrotic and apoptotic cells.
CRP is a member of the class of acute phase reactants, as its levels rise dramatically during inflammatory processes occurring in the body. This increment is due to a rise in the plasma concentration of IL 6, which is produced predominantly by macrophages as well as adipocytes. CRP binds to phosphocholine on microbes. It is thought to assist in complement binding to foreign and damaged cells and enhances phagocytosis by macrophages (opsonin mediated phagocytosis), which express a receptor for CRP. It is also believed to play another important role in innate immunity as an early defense system against infections.
CRP rises up to 50,000-fold in acute inflammation, such as infection. It rises above normal limits within 6 hours and peaks at 48 hours. Its half-life is constant and therefore, its level is mainly determined by the rate of production (hence the severity of the precipitating cause).
CRP levels also tend not to be elevated in systemic lupus erythematosus (SLE) unless serositis or synovitis is present. Elevations of CRP in the absence of clinically significant inflammation can occur in renal failure. CRP level is an independent risk factor for atherosclerotic disease. Patients with high CRP concentrations are more likely to develop stroke, myocardial infarction, and severe peripheral vascular disease.
Recent research suggests that patients with elevated basal levels of CRP are at an increased risk of diabetes, hypertension, and cardiovascular disease. A study of over 700 nurses showed that those in the highest quartile of trans fat consumption had blood levels of CRP that were 73% higher than those in the lowest quartile. Although one group of researchers indicated that CRP may be only a moderate risk factor for cardiovascular disease, this study (known as the Reykjavik Study) was found to have some problems for this type of analysis related to the characteristics of the population studied, and there was an extremely long follow-up time, which may have attenuated the association between CRP and future outcomes. Others have shown that CRP can exacerbate ischemic necrosis in a complement -dependent fashion, and that CRP inhibition can be a safe and effective therapy for myocardial and cerebral infarcts ; so far, this has been demonstrated in animal models only.
CRP protein is used mainly as a marker of inflammation. Apart from liver failure, there are few known factors that interfere with CRP production. Measuring and charting CRP values can prove useful in determining disease progress or the effectiveness of treatments. Blood, usually collected in a serum separating tube, is analyzed in a medical laboratory or at the point of care. Various analytical methods are available for CRP determination, such as ELISA, immunoturbidimetry, rapid immunodiffusion and visual agglutination.
A high sensitivity CRP (hs CRP) test measures low levels of CRP using laser nephelometry. The test gives results in 25 minutes with sensitivity down to 0.04 mg/L. Normal concentration in healthy human serum is usually lower than 10 mg/L, slightly increasing with aging. Higher levels are found in late pregnant women, mild inflammation and viral infections (10–40 mg/L), 21active inflammation, and bacterial infection (40–200 mg/L); and severe bacterial infections and burns (> 200 mg/L).
CRP is a more sensitive and accurate reflection of the acute phase response than the erythrocyte sedimentation rate (ESR). The half-life of CRP is constant. Therefore, CRP level is mainly determined by the rate of production (and hence the severity of the precipitating cause). In the first 24 hours, ESR may be normal and CRP elevated. CRP returns to normal more quickly than ESR in response to therapy.
Arterial damage results from white blood cell invasion and inflammation within the wall. CRP is a general marker for inflammation and infection, so it can be used as a very rough proxy for heart disease risk. Since many things can cause elevated CRP, this is not a very specific prognostic indicator. Nevertheless, a level above 2.4 mg/L has been associated with a doubled risk of a coronary event compared to levels below 1 mg/L. However, in the study group in this case consisted of patients who had been diagnosed with unstable angina pectoris, whether elevated CRP has any predictive value of acute coronary events in the general population of all age ranges remains unclear.
 
Immunoglobulin Light Chain
Light chains are of two types, kappa (κ) and lambda (λ), and in any given antibody molecule only one type occurs. Approximately twice as many κ as λ molecules are produced in humans, but in other mammals, this ratio can vary. Each free light chain (FLC) molecule contains approximately 220 amino acids in a single polypeptide chain that is folded to form the constant and variable region domains (Fig. 8).
Domains are constructed from two β sheets. These are elements of protein structure made up of strands of the polypeptide chain (β strands) packed together in a particular shape. The sheets are linked to a disulfide bridge and together form a roughly barrel-shaped structure known as a β barrel.
Fig. 8: An antibody molecule showing the heavy and light chain structure, together with kappa and lambda free light chains
22
Genes, which encode antibodies are assembled in B lymphocytes by joining multiple gene segments, which are far apart in germline DNA. The light chain variable domain is constructed from V and J gene segments, whilst the constant domain is encoded by a separate C gene segment.
There are multiple copies of the gene segments in germline DNA, and a random selection of individual V, J and C genes contributes to the diversity of immunoglobulin light chains. The constant domains of light chains show little amino acid sequence variation. The human genome contains a single κ constant gene for which three serologically defined allotypes have been defined, and designated Km1, Km2 and Km3. These allotypes define three Km alleles that differ in two amino acids. The genome also contains a variable number of λ constant genes, giving rise to multiple λ chain isotypes. These λ isotypes can be distinguished serologically through the expression of Mcg, Kern, and Oz markers. For example, the protein product of the IGLC1 gene is Mcg+Kern+Oz-. There is no evidence that these variants affect FLC measurements using free light assays, which are based on polyclonal antisera. In contrast, the variable domains of light chains exhibit huge structural diversity, particularly in association with the antigen binding amino acids. In addition, the first 23 amino acids of the first variable domain framework region have a limited number of variations known as subgroups. Using monoclonal antibodies, four κ (Vκ1–Vκ4) and six λ subgroups (Vλ1–Vλ6) can be identified. The specific subgroup structures influence the potential of the FLCs to polymerize such that amyloid light chain (AL) amyloidosis is associated with Vλ6 and light chain deposition disease (LCDD) with Vκ1 and Vκ4.
Kappa FLC molecules (chromosome 2) are constructed from approximately 40 functional Vκ gene segments, five Jκ gene segments and a single Cκ gene. Lambda FLC molecules (chromosome 22) are constructed from approximately 30 Vλ gene segments and four (or more) pairs of functional Jλ gene segments and Cλ genes. FLCs are incorporated into immunoglobulin molecules during B lymphocyte development and are expressed initially on the surface of pre-B cells. Production of FLCs occurs throughout the rest of B cell development and in plasma cells, where secretion is highest. Tumors associated with the different stages of B cell maturation will secrete monoclonal FLCs into the serum where they may be detected by FLC immunoassays (Fig. 9).
In normal individuals, approximately 500 mg of FLCs are produced each day from bone marrow and lymph node cells. The molecules enter the blood and are rapidly partitioned between the intravascular and extravascular compartments. The normal plasma cell content of the bone marrow is about 1%, whereas in multiple myeloma this can rise to over 90%. The bone marrow may contain 5–10% plasma cells in chronic infections and autoimmune diseases, and this is associated with hypergammaglobulinemia and corresponding increases in polyclonal serum free light chain (sFLC) concentrations. Bone marrow identification of monoclonal plasma cells by histology or flow cytometery is an essential part of multiple myeloma (MM) diagnosis and is frequently based on identifying intracellular κ and λ by direct immunofluorescence techniques.23
Fig. 9: Development of the B cell lineage and associated diseases
Plasma cells produce one of five heavy chain types, together with κ or λ molecules. There is approximately 40% excess FLC production over heavy chain synthesis, to allow proper conformation of the intact immunoglobulin molecules. As mentioned previously, there are twice as many κ producing plasma cells as λ producing plasma cells. Kappa FLCs are normally monomeric, while λ FLCs tend to be dimeric, joined by disulfide bonds; however higher polymeric forms of both FLCs may occur. In normal individuals, sFLCs are rapidly cleared and metabolized by the kidneys depending upon their molecular size. Monomeric FLCs, characteristically κ, are cleared in 2–4 hours at 40% of the glomerular filtration rate. Dimeric FLCs, typically λ, are cleared in 3–6 hours at 20% of the glomerular filtration rate, while larger polymers are cleared more slowly. Removal may be prolonged to 2–3 days in MM patients in complete renal failure. In contrast, IgG has a half- life of 21 days with minimal renal clearance.24
 
Bence Jones Protein
Considered as the first tumor marker, it is a monoclonal globulin protein or immunoglobulin light chain found in the blood or urine, with a molecular weight of 22–24 k D. It is excreted in the urine of myeloma patients. The presence of protein is a sign that there are higher than normal levels of protein in the blood, as in the case of myeloma where there is an overproduction of immunoglobulin protein. When Bence Jones proteins are in the urine, they can accumulate and cause kidney damage.
Detection of Bence Jones protein may be suggestive of MM or WM. Bence Jones proteins are particularly diagnostic of multiple myeloma in the context of end-organ manifestations, such as renal failure, lytic (or “punched out”) bone lesions, anemia, or large numbers of plasma cells in the bone marrow of patients. Bence Jones proteins are present in two-third of MM cases.
The proteins are immunoglobulin light chains (paraproteins) and are produced by neoplastic plasma cells. They can be κ (most of the time) or λ. The light chains can be immunoglobulin fragments or single homogeneous immunoglobulins. They are found in urine due to the kidneys’ decreased filtration capabilities due to renal failure, sometimes induced by hypercalcemia from the calcium released as the bones are destroyed or from the light chains themselves. The light chains have historically been detected by heating and now by electrophoresis of concentrated urine. More recently serum free light chain assays have been utilized in a number of published studies, which have indicated superiority over the urine tests, particularly for patients producing low levels of monoclonal FLCs, as seen in nonsecretory MM and AL amyloidosis. This is primarily because of the reabsorption of FLCs in the kidneys, creating a “threshold” of light chain production which must be exceeded before measurable quantities overflow into the urine. As such, urinalysis is a fickle witness to changing FLC production.
 
URINARY PROTEIN ELECTROPHORESIS
The association of the proteinuria with abnormality of the kidney has been clearly demonstrated by Richard Bright around 150 years ago. The role of the kidney in the storage and metabolism of the plasma proteins has been studied many times in the past, but it is in the last two decades that the subject has been the focus of numerous studies.
The interest in the study of proteinuria is derived by the frequency of appearance of this sign, present in almost all kidney diseases, by the development of biochemical methods for the qualitative and quantitative study of urinary proteins, and a more precise knowledge of the physiological processes of glomerular filtration and tubular reabsorption of the proteins in normal and pathological conditions (Fig. 10).
The presence of proteinuria does not necessarily imply the existence of a renal pathology, even normal subjects have “physiological” proteinuria (Box 1), and instead, there are the qualified electrophoretic studies to define a proteinuria as “pathological”, highlighting the presence of plasma originating proteins in the urine that under normal conditions do not exceed the glomerular barrier of proteins present only when the tubule does not 25carry out its function of reabsorption of abnormal plasmatic proteins that pass in the urine, and of proteins secreted by the kidney and urinary tract.
Fig. 10: The nephron consists of two parts—glomerulus and the tubule
 
Glomerular Function
Four main factors intervene in the determination of the glomerular filtration of the proteins:
  1. The selective permeability: molecular weight and form
  2. The presence of anionic structure with the function of electrostatic repulsion
  3. The plasmatic concentration
  4. The local hemodynamic conditions.
 
Tubular Function
 
Mechanisms of Reabsorption
All proteins with a molecular weight less that 40 D do not encounter, almost, difficulty in being filtered at the glomerular level, and they are over 90% reabsorbed in the proximal tubule.26
 
Clinical Aspects
From the semiological viewpoint, the proteinuria may be:
  • Transitory
  • Intermittent
  • Persistent.
 
Transitory Proteinuria
It consists of the exhibition of occasional proteinuria with pathological values, usually moderate that do not repeat over time in subsequent checks and has a benign type character. With the end of functional proteinuria, meaning the proteinuria that independently from pathological kidney states accompanies several clinical conditions characterized by states of fever to intense and prolonged exercise to conditions of physical and emotional stress, such as surgical interventions or the exposure to cold. This proteinuria is defined as transitory type and resolves together at the recovery of the physical condition altering it was not the origin.
 
Intermittent Proteinuria
These proteinurias are characterized, as their definition indicates, by the appearance of pathological proteins over time intervals and in episodes in which the proteinuria re-enters into the range of physiological values. These proteinurias may have completely random and irregular courses, or show a regular recurrence such as in orthostatic type proteinuria, in addition to normal values in the samples taken in the morning, following a period of supine rest, which presents pathological values in serotin samples, after the common orthostatic activity. The intermittent proteinurias are of glomerular type, although usually quantitatively small, they deserve attention because they can be indexes of incipient renal suffering. The verification of finding an intermittent proteinuria requires the doctor to deepen the investigations in order to be able to resolve the potential doubt of a renal lesion that, as we have said, may be the cause. The typology of the intermittent proteinuria is often a prerogative of the child and adolescent age, where it is fundamental to exclude the possibility of silent Streptococcus infections.
 
Persistent Proteinuria
It can be distinguished in the prerenal, renal, and postrenal forms. The prerenal proteinurias are the once from “overflow”, which are not the expression of renal disease, but proteins physiologically filtered by glomerulus that are present in an increased concentration in the plasma for various pathological contingencies, exceeding the normal threshold of tubular reabsorption paradigmatic expression of this type of proteinuria and the Bence Jones proteinuria. The renal proteinurias are those already described in the etiopathogenetic classification as glomerulary, tubulary, 27and mixed. The postrenal proteinurias are due to lesions, which often refer to an inflammatory nature of the tissues of the kidney pathways, such as renal pelvis, ureter, bladder, prostate, and genital organs. Sometimes, these proteinurias have a composition, which allows the identification of the presence, of specific proteins such as the presence of enzymes or the Tamm Horsfall protein.
 
Pathogenetic Classification of the Proteinurias
The complexity of the mechanisms underlying the determinism of proteinuria is reflected in the complexity of the interpretative diagnostic process of the proteinuria frameworks found in clinical practice on the basis of the probable origin of urinary proteins (Tables 1 to 5).
 
Proteinurias of Plasmatic Derivation
Table 1   Pathogenetic classification of proteinuriqs of the plasmatic derivation (renal glomerular)
Type
Selective glomerular proteinuria
Glomerular proteinuria
Non selective
Cause
Alterations of basal membrane load for loss of negative charges
Alterations of the glomerular barrier structure for an increase
and/or enlargement of the pores
Protein classes
Albumin and transferrin
Albumin transferrin, and Immunoglobulins
Molecular weight
68,000–77,000 D
68,000–500,000 D
Examples of correlated pathologies
Minimal change Nephropathy
Focal glomerular sclerosis, glomerule membranous
Proliferative glomerulonephritis
Advanced diabetes
Amyloidosis
28
Table 2   Pathogenetic classification of proteinurias of the plasmatic deriviation (renal tubular)
Type
Complete tubular proteinuria
Incomplete tubular proteinuria
Cause
Defect of reabsorption of the proteins at low molecular weight, normally filtered
Defect of reabsorption of the proteins at low molecular weight, normally filtered
Protein classes
Hormones enzymes microglobulins, peptides, and others
Hormones enzymes, microglobulins, peptides, and others
Molecular weight
15,000–70.000 D
40,000–70,000 D
Examples of correlated pathologies
Interstitial nephritis Nephrotoxicity from heavy metals Nephrotoxicity from drugs
Rejection of kidney transplant
Diabetic glomerulosclerosis Hypertensive glomerulosclerosis
Table 3   Pathogenetic classification of proteinurias of the plasmatic derivation (prerenal from overflow)
Type
From overflow
From overflow
From overflow
Cause
From tissular destruction
From over production
From secretion
Protein classes
Myoglobin haemoglobin, and lysozyme
Light chains and Lysozyme fragments
Amylase proteins at low PM “histuria”
Molecular weight
Respectively
17,000 D
16,800 D monomer
14,500 D
Respectively
22,000 D monomer
44,000 D dimer
14,500 D
Respectively
50,000 D
?
Examples of correlated pathologies
Respectively Rhabdomyolysis Myocardial infarction Crash syndromes
Hemolytic autoimmune diseases
myelomonocytic Leukemias
Monoblastic leukemias
Respectively Myeloma Lymphoma
Waldenstrom disease
myelomonocytic leukemias Monoblastic leukemias
Respectively
Pancreatitis
?
29
 
Proteinurias of Tubular Derivation
Table 4   Pathogenetic classification of proteinurias of the tubular derivation (nephrogenetic)
Type
Nephrogenetic proteinurias
Nephrogenetic proteinurias
Cause
Local secretion
Exfoliation or destruction of renal tissue
Protein classes
Uromucoid or protein
Tamm Horsfall
secretory IgA
Enzymes from tubular cells
Renal antigens
Molecular weight
Respectively
80,000–>7 x 106 D
320,000 D
Respectively
90,000 D
?
Examples of correlated pathologies
Physiological situation
Respectively Acute tubulopathy Chronic tubulopathy Transplant rejection
 
Post renal Proteinurias (Table 5)
Table 5   Pathogenetic classification of the postrenal proteinurias
Type
Post renal proteinurias
Cause
Local bleeding
Local secretion of immunoglobulins
Chyluria
Protein classes
Plasmatic proteins
Immunoglobulin
Lipoproteins
Molecular weight
60,000–500,000 D
> 150,000 D
?
Examples of correlated pathologies
Calculosis Inflammation Neoplasms
Inflammations
Obstruction from neoplasm
30

Serum Protein Electrophoresis: IndicationsCHAPTER 2

 
INTRODUCTION
Serum protein electrophoresis is commonly performed when multiple myeloma (MM) is suspected (Table 1). MM is a disease with many faces. It usually presents in old age but may occur in youth. Bone pain and fractures are characteristics, yet soft tissue involvement by plasmacytomas may also occur. Patients may die within weeks of presentation, while others “smolder” for years. Patients may develop renal failure, acute and chronic infections or AL amyloidosis, and many will require stem cell transplantation or intensive chemotherapy. Consequently, many specialists, including hematologists, nephrologists, immunologists, orthopedic surgeons, and chemical pathologists become involved in disease management. Furthermore, the prevalence of MM is increasing due to a slowly rising incidence and a longer life expectancy.
Table 1   Indications for serum protein electrophoresis
Suspected multiple myeloma, Waldenström's macroglobulinemia, or primary amyloidosis or related disorder
Unexplained peripheral neuropathy (not attributed to long-standing diabetes mellitus, toxin exposure, chemotherapy, etc.)
New onset anemia associated with renal failure or insufficiency and bone pain
Back pain in which multiple myeloma is suspected
Hypercalcemia attributed to possible malignancy (e.g. associated weight loss, fatigue, bone pain, abnormal bleeding, etc.)
Rouleaux formations noted on peripheral blood smear
Renal insufficiency with associated serum protein elevation
Unexplained pathologic fracture or lytic lesion identified on radiograph
Bence Jones proteinuria
32
Despite the complexity of this disease, one feature has been a great lighthouse in the fog, alerting the unwary to the diagnosis and guiding the hand of management—the presence of monoclonal immunoglobulins. Produced in excess and in a variety of shapes and sizes, these molecules have been linked to MM since they were first identified by Henry Bence Jones over 150 years ago.
On Friday, October 30, 1845, the 53-year-old Dr William MacIntyre, physician to the Western General Dispensary, St. Marylebone, London, left his rooms in Harley Street. He had been called to see Mr Thomas Alexander McBean, a 45-year-old, highly respectable grocer, who had severe bone pain and fractures. He had been under the care of his general practitioner, Dr Thomas Watson, for several months. Upon examination of the patient, William MacIntyre noted the presence of edema. Considering the possibility of nephrosis, he tested the urine for albumin. To his consternation, the albuminous protein precipitate found on warming the urine, uncharacteristically, redissolved when heated to 75°C.
Both Dr MacIntyre and Dr Watson then sent urine samples to the chemical pathologist at St. George's Hospital. A note accompanying the urine sent by Dr Watson read as follows:
“Dear Dr Bence Jones, The tube contains urine of very high specific gravity. When boiled it becomes highly opaque. On the addition of nitric acid, it effervesces, assumes a reddish hue, and becomes quite clear; but as it cools assumes the consistence and appearance which you see.
Henry Bence Jones (December 31, 1813—April 20, 1873) was an English physician and chemist . He entered Harrow in 1827 and then went up to Trinity College, Cambridge in 1832, obtaining his degree in 1836. He initially worked for an apothecary but subsequently (1838) enrolled to study medicine at St George's Hospital , and in 1839, chemistry at University College, London . In 1841, he went to Giessen in Germany to work at chemistry with Liebig . On his return, he took a post at St George's hospital and, after being promoted to assistant physician, was elected in 1846 to full physician, resigning on health grounds in 1862. In 1847, he described the Bence Jones protein , a globulin protein found in blood and urine, suggestive of multiple myeloma or Waldenström's macroglobulinemia . Besides becoming a fellow, and afterwards senior censor, of the Royal College of Physicians , and a fellow of the Royal Society , he held the post of secretary to the Royal Institution for many years. He died in London on April 20, 1873. He had married his second cousin and had seven children.
33
Heat reliquefies it. What is it?”
Over the next 2 months, the patient deteriorated, became emaciated, weak, and was racked with pain. He eventually died on January 1, 1846, in full possession of his mental faculties. Dr MacIntyre subsequently published the postmortem examination and the description of the peculiar urine in 1850. Unfortunately, for him, Henry Bence Jones had already described the patient's urinary findings in two, single-author articles, one of which was published in The Lancet, in 1847. He considered the protein to be a “hydrated deutoxide of albumen”. He wisely commented:
I need hardly remark on the importance of seeking for this oxide of albumin in other cases of mollities ossium.
Henry Bence Jones's reputation was assured, while the contributions of his colleagues were consigned to the footnotes of history.
For all the apparent injustice to his colleague (William MacIntyre), Henry Bence Jones achieved much else in his career. He published over 40 papers and became rich and famous based on his clinical practice, lecturing, and original observations and was elected to fellowship of The Royal Society at the tender age of 33. Florence Nightingale once described him as “the best chemical doctor in London”. Surprisingly, there was no mention of Bence Jones protein in his obituary and the eponym (and the hyphen in his name) was not used until after his death.
By 1909, over 40 cases of Bence Jones proteinuria had been reported, and the protein was thought to originate in bone marrow plasma cells that were first identified by Waldeyer in 1875. In 1922, Henry Bayne Jones and Wilson characterized two types of Bence Jones protein by observing precipitation reactions using antisera made by immunizing rabbits with the urine of several patients. The proteins were classified as group I and group II types. However, it was not until 1956 that Korngold and Lapiri, using the Ouchterlony technique, showed that antisera raised against the different groups also reacted with myeloma proteins. As a tribute to their observations the two types of Bence Jones protein were designated kappa (κ) and lambda (λ). Edelman and Gally, in 1962, subsequently showed that free light chains (FLCs) prepared from IgG monoclonal proteins were the same as Bence Jones protein. It had taken 117 years (Fig. 1) from the original observation for the function of Bence Jones protein to be finally determined. Remarkably, the format of the urine test had remained unchanged for a similar period.
 
MONOCLONAL GAMMOPATHY
Monoclonal gammopathy is a condition of an increased production of one type of immunoglobulin by a single clone of cells. The abnormal protein produced is called paraprotein or M component, and it is categorized according to the type of monoclonal protein found in blood:
  • Light chains only (or Bence Jones protein). This may be associated with MM or AL amyloidosis
  • Heavy chains only (also known as “ heavy chain disease ”)34
    Fig. 1: Bence Jones and the history of free light chains
  • Whole immunoglobulins. In this case, the paraprotein goes under the name of “M protein” (“M” for monoclonal). If immunoglobulins tend to precipitate within blood vessels with cold, that phenomenon takes the name of cryoglobulinemia.
The three types of paraproteins may occur alone or in combination in a given individual. Note that, while most heavy chains or whole immunoglobulins remain within blood vessels, light chains frequently escape and are excreted by the kidneys into urine, where they take the name of Bence Jones protein. It is also possible for paraproteins (usually whole immunoglobulins) to form polymers by aggregating with each other; this takes the name of macroglobulinemia and may lead to further complications. For example, certain macroglobulins tend to precipitate within blood vessel with cold, a phenomenon known as c ryoglobulinemia. Others may make blood too viscous to flow smoothly (usually with IgM macroglobulins), a phenomenon known as Waldenström macroglobulinemia.
The globulins separate out into three regions on the electrophoretic gel that are: (i) the α band, (ii) the β band, and (iii) the γ band.
  1. The α band can be separated into two components: (i) α1 and (ii) α2. The α1 region consists mostly of α1 antitrypsin and α1 acid glycoprotein. The α2 region is mostly haptoglobin, α2 macroglobulin, α2 antiplasmin, and ceruloplasmin.
  2. The β band consists of transferrin, low density lipoproteins and complement system proteins.35
  3. The γ band is where the immunoglobulins appear, which is why they are also known as gammaglobulins. The majority of paraproteins appear in this band.
 
Malignant Monoclonal Gammopathies
  1. MM (IgG, IgA, IgD, IgE, and free κ or λ light chains):
    • Overt MM
    • SMM
    • Plasma cell leukemia
    • NSMM
    • Polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes (POEMS); osteosclerotic myeloma.
  2. Plasmacytoma:
    • Solitary plasmacytoma of bone
    • Extramedullary plasmacytoma
    • Malignant lymphoproliferative disorders
    • Waldenström's macroglobulinemia
    • Malignant lymphoma
    • Chronic lymphocytic leukemia or lymphoproliferative disorders
    • Heavy chain diseases
    • γ heavy chain disease
    • α heavy chain disease
    • μ heavy chain disease
    • Amyloidosis
    • Primary amyloidosis
    • With MM (secondary, localized and familial amyloidosis have no monoclonal protein).
 
Nonmalignant Disorders Associated Rarely with Monoclonal Proteins (Usually MGUS)
  • Dermatological diseases
  • Lichen myxoedematosus (IgG1), scleroderma, pyoderma gangrenosum, necrobiotic xanthogranuloma, discoid lupus erythematosus, psoriasis, cutaneous lymphoma
  • Immunosuppression
  • AIDS and HIV infection, renal transplantation, bone marrow transplantation
  • Liver diseases
  • Chronic hepatitis, cirrhosis, primary biliary cirrhosis
  • Miscellaneous
  • Rheumatoid arthritis, inflammatory seronegative polyarthritis, polymyositis (IgGκ), polymyalgia rheumatica, myasthenia gravis, angioneurotic edema (C1 inactivator deficiency).
Pathologically, the lesion in MGUS is in fact very similar to that in MM. There is a predominance of clonal plasma cells in the bone marrow with an abnormal immunophenotype (CD38 + CD56 + CD19 −) mixed in with cells of a normal phenotype (CD38+ CD56− CD19+); in MGUS, on average, more than 363% of the clonal plasma cells have the normal phenotype, whereas in MM, less than 3% of the cells have the normal phenotype. What causes MGUS to transform into MM is as yet unknown. At the Mayo Clinic, MGUS transformed into MM or similar myeloproliferative disorder at the rate of about 1–2% a year, or at 17%, 34% and 39% in 10, 20 and 25 years, respectively, of follow-up—among surviving patients. However, because they were elderly, most patients with MGUS died of something else and did not go on to develop MM. When this was taken into account, only 11.2% developed myeloproliferative disorders.
Kyle studied the prevalence of myeloma in the population as a whole (not clinic patients) in Olmsted County, Minnesota, USA. They found that the prevalence of MGUS was 3.2% in people above 50 years of age, with a slight male predominance (4% vs 2.7%). Prevalence increased with age: people over 70 years up to 5.3% had MGUS, while in the over 85 years age group the prevalence was 7.5%. In the majority of cases (63.5%), the paraprotein level was less than 1/dL, while only a very small group had levels over 2/dL. A study of monoclonal protein levels conducted in Ghana showed a prevalence of MGUS of approximately 5.9% in African men over the age of 50 years.
In 2009, prospective data demonstrated that all or almost all cases of MM are preceded by MGUS. In addition to MM, MGUS may also progress to Waldenström's macroglobulinemia, primary amyloidosis, B cell lymphoma or chronic lymphocytic leukemia. The protein electrophoresis test should be repeated annually, and if there is any concern for a rise in the level of monoclonal protein, then prompt referral to a hematologist is required. The hematologist, when first evaluating a case of MGUS, will usually perform a skeletal survey (X-rays of the proximal skeleton), check the blood for hypercalcemia and deterioration in renal function, check the urine for Bence Jones protein and perform a bone marrow biopsy. If none of these tests are abnormal, a patient with MGUS is followed up once every 6 months to a year with a blood test (serum protein electrophoresis).
 
Polyclonal Gammopathy
Polyclonal gammopathy reflects the presence of a diffuse hypergamma-globulinemia in which all immunoglobulin classes are proportionally increased.
A hypergammaglobulinemia resulting from an increased production of several different immunoglobulins and usually attributable to persistent, high level exposure to antigens occurs in a wide variety of infectious, inflammatory and immune mediated diseases. Chronic infections, autoimmune diseases, and many tumors cause increases in polyclonal immunoglobulins and presumably polyclonal FLC concentrations. Skin, pulmonary, and gut diseases are more likely to cause increases in IgA concentrations while systemic infections will increase all immunoglobulins but particularly IgG.
  • Connective tissue diseases: Sjögren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, mixed connective tissue disease, overlap syndrome, juvenile rheumatoid arthritis, progressive systemic sclerosis, ankylosing spondylitis, fibrosing alveolitis, CREST syndrome, temporal 37arteritis, Raynaud's phenomenon, cutaneous vasculitis, familial mediterranean fever, eosinophilic fasciitis, and inclusion body myositis
  • Liver diseases (61%): Autoimmune hepatitis, viral hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, cryptogenic cirrhosis, primary hemochromatosis, ethanol induced liver injury, and α1 antitrypsin deficiency
  • Chronic infections (6%): Subacute bacterial endocarditis, renal abscess, cystic fibrosis, Whipple's disease, brucellosis, Lyme disease, malaria, worm infestations, tropical splenomegaly syndrome, Mycobacterium tuberculosis, Mycobacterium leprae, Leishmania organisms, Trypanosoma cruzi, Toxocara canis, HIV1, varicella and vaccinia
  • Lymphoproliferative disorders (5%): Pseudolymphoma, Kikuchi disease, malignant lymphoma, Castleman disease, angioimmunoblastic lymphadenopathy with dysproteinemia, large granular lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, plasma cell leukemia, histiocytosis X, sinus histiocytosis with massive lymphadenopathy, cutaneous eruptive histiocytoma, intracranial plasma cell granulomata, systemic cutaneous plasmacytosis, proteinaceous lymphadenopathy with hypergammaglobulinemia, chronic active EBV infection syndrome, and severe autoimmune lymphoproliferative syndrome
  • Other hematological conditions: Myelodysplastic syndromes, idiopathic neutropenia, idiopathic thrombocytopenic purpura, severe hemophilia A, thalassemia major, sickle-cell anemia, benign hypergammaglobulinemia purpura of Waldenström, cryoglobulinemia, and Fanconi anemia
  • Nonhematological malignancies (3%): Gastric carcinoma, lung cancer, hepatocellular carcinoma, renal cell carcinoma, ovarian cancer, and chondrosarcoma
  • Neurological conditions: Acquired chronic dysimmune demyelinating polyneuropathy, HTLV-1 associated myelopathy, chronic progressive sensory ataxic neuropathy, pure motor neuron disease and plasma cell dyscrasia, and microangiopathy of vasa nervorum in dysglobulinemic neuropathy
  • Diseases with associated immune system abnormalities: Graves’ disease, chronic ulcerative colitis, chronic autoimmune pancreatitis, sarcoidosis, syndrome of IgG2 subclass deficiency, hyper IgE syndrome, hyperimmunoglobulinemia D, and periodic fever syndrome
  • Drugs: Aminophenazone, asparaginase, ethotoin, hydralazine hydrochloride, mephenytoin, methadone, oral contraceptives, phenylbutazone and phenytoin
  • Miscellaneous conditions: Gaucher's disease, Ménière's disease, cardiac myxoma, asbestos exposure, cryptogenic organizing pneumonitis, lymphoid interstitial pneumonia, distal-renal-tubular acidosis, and hyperimmunization.
 
Biclonal Gammopathy
Biclonal gammopathy is a primary disturbance in immunoglobulin synthesis characterized by serum containing two distinct monoclonal immunoglobulins. The presence of two clonal expansions—IgG > IgA > IgM—found 38in the same patient. The biclonal gammopathies were associated with malignancies—on bronchial small cell carcinoma and the other colon cancer. Both patients died soon after the discovery of their respective tumors.
 
Triclonal Gammopathy
Triclonal gammopathy is an immunoglobulin abnormality in which three discrete monoclonal subpopulations of immunoglobulin molecules are present in a patient's serum. Monoclonal and biclonal gammopathies have been studied extensively, but relatively little is known about the much rarer triclonal gammopathies.
The triclonal gammopathies had the following associations: non-Hodgkin's large cell lymphoma, breast cancer with possible lymphoma and severe inflammatory pathologies that we were unable to further identify; patients in this group died. These data are in light of the rarity of reported cases of biclonal and triclonal gammopathies and the controversies surrounding their possible association with a malignant pathology.
 
Oligoclonal Gammopathy
Oligoclonal gammopathy bands are the bands of immunoglobulins, which are seen when a patient's blood serum, gained from blood plasma, or cerebrospinal fluid (CSF), is analyzed.
The following two methods of analysis are possible:
  1. Protein electrophoresis.
  2. The combination of isoelectric focusing/silver staining.
Each of the two to five oligoclonal bands seen by protein electrophoresis represents proteins (or protein fragments) secreted by plasma cells, although why exactly these bands are present, and which proteins these bands represent, has not yet been elucidated. The presence of oligoclonal bands in CSF combined with their absence in blood serum often indicates that immunoglobulins are produced in central nervous system (CNS). Therefore, it is normal to subtract bands in serum from bands in CSF when investigating CNS diseases.
Oligoclonal bands are an important indicator in the diagnosis of multiple sclerosis. Approximately 79–90% of all patients with multiple sclerosis have permanently observable oligoclonal bands. The presence of one band (a monoclonal band) may be considered serious, such as lymphoproliferative disease, or may simply be normal—it must be interpreted in the context of each specific patient. More bands may reflect the presence of a disease. The bands tend to disappear from the CSF as a person recovers from the neurological disease.
Oligoclonal bands are also found in:
  • Multiple sclerosis
  • Lyme disease
  • Devic's disease
  • Systemic lupus erythematosus
  • Neurosarcoidosis39
  • Subacute sclerosing panencephalitis
  • Subarachnoid hemorrhage
  • Syphilis
  • Primary CNS lymphoma
  • Sjögren's Syndrome
  • Guillain-Barré syndrome.
40

Serum Protein Electrophoresis: Clinical SignificanceCHAPTER 3

 
Introduction
Protein electrophoresis is a well-established technique routinely used in clinical laboratories for screening of serum and some other fluids for protein abnormalities. It is based on the principles of zone electrophoresis performed on a suitable support medium. Agarose has been developed into a versatile and effective support medium. For routine diagnostic applications, serum proteins separate into five major fractions, primarily according to their charge at a given pH: albumin, alpha 1 (α1) globulins, α2 globulins, beta (β) globulins, and gamma (γ) globulins. Each zone contains one or more serum proteins. The urine protein patterns resemble those of serum. However, the relative intensities of the fractions or their presence may vary greatly depending on the filtration capability of the kidney.
Serum protein electrophoresis is used to identify patients with multiple myeloma (MM) and other serum protein disorders. Serum protein levels display reasonably predictable changes in response to acute inflammation, malignancy, trauma, necrosis, infarction, burns, and chemical injury. A homogeneous spike like peak in a focal region of the γ globulin zone indicates a monoclonal gammopathy.
Monoclonal gammopathies are associated with a clonal process, which is malignant or potentially malignant, including MM, Waldenstrom's macroglobulinemia (WM), solitary plasmacytoma, smoldering MM, monoclonal gammopathy of undetermined significance (MGUS), plasma cell leukemia, heavy-chain disease, and amyloidosis. The quantity of monoclonal protein (M protein), the results of bone marrow biopsy, and other characteristics can help differentiate MM from the other causes of monoclonal gammopathy. In contrast, polyclonal gammopathies may be caused by any reactive or inflammatory process.42
Plasma protein levels display reasonably predictable changes in response to acute inflammation, Malignancy, trauma, necrosis, infarction and burns and chemical injury. The so-called “acute reaction protein pattern” involves increases in fibrinogen, α1 antitrypsin, haptoglobin, ceruloplasmin, C-reactive protein (CRP), the C3 portion of complement, and α1 acid glycoprotein. Often, there are associated decreases in the patterns of acute reaction proteins found on serum protein electrophoresis, along with associated conditions or disorders. In the interpretation of serum protein electrophoresis, most attention focuses on the γ region that is composed predominantly of antibodies of the immunoglobulin G (IgG) type.
The γ globulin zone is decreased in hypogammaglobulinemia and agammaglobulinemia. Diseases that produce an increase in the γ globulin level include Hodgkin's disease, malignant lymphoma, chronic lymphocytic leukemia, granulomatous diseases, connective tissue diseases, liver diseases, MM, WM and amyloidosis. Although many conditions can cause an increase in the γ region, several disease states cause a homogeneous spike like peak in a focal region of the γ globulin zone.
 
MONOCLONAL VERSUS POLYCLONAL GAMMOPATHIES
It is extremely important to differentiate monoclonal from polyclonal gammopathies. Monoclonal gammopathies are associated with a clonal process that is malignant or potentially malignant. In contrast, polyclonal gammopathies may be caused by any reactive or inflammatory process, and they usually are associated with nonmalignant conditions. An M protein is characterized by the presence of a sharp, well-defined band with a single heavy chain and a similar band with a kappa (κ) or lambda (λ) light chain. A polyclonal gammopathy is characterized by a broad diffuse band with one or more heavy chains and κ and λ light chains. Once a monoclonal gammopathy is identified by serum protein electrophoresis, MM must be differentiated from other causes of this type of gammopathy. Among these other causes are WM, solitary plasmacytoma, smoldering MM, MGUS, plasma cell leukemia, heavy chain disease, and amyloidosis.
The quantity of M protein can help differentiate MM from MGUS. Definitive diagnosis of MM requires 10–15% plasma cell involvement as determined by bone marrow biopsy. Characteristic differentiating features of the monoclonal gammopathies are listed in (Table 1).
In some patients with a plasma cell dyscrasia, serum protein electrophoresis may be normal, because the complete monoclonal immunoglobulin is absent or is present at a very low level. In one series, serum protein electrophoresis showed a spike or localized band in only 82% of patients with MM.
The remainder had hypogammaglobulinemia or a normal appearing pattern. Consequently, urine protein electrophoresis is recommended in all patients suspected of having a plasma cell dyscrasia.
An additional point to consider is the size of the M protein spike. Although this spike is usually greater than 3 g/dL in patients with MM, up to one-fifth of patients with this tumor may have an M protein spike of less than 1 g/dL. Hypogammaglobulinemia on serum protein electrophoresis occurs in about 4310% of patients with MM who do not have a serum M protein spike.
Table 1   Characteristic features of monoclonal gammopathies
Disease
Distinctive features
Multiple myeloma
M protein appears as a narrow spike in the γ, β, or α2 regions
M protein level is usually greater than 3 g/dL
Skeletal lesions (e.g. lytic lesions, diffuse osteopenia and vertebral compression fractures) are present in 80% of patients
Diagnosis requires 10–15% plasma cell involvement on bone marrow biopsy
Anemia, pancytopenia, hypercalcemia, and renal disease may be present
Monoclonal gammopathy of undetermined significance
M protein level is < 3 g/dL
There is < 10% plasma cell involvement on bone marrow biopsy
Affected patients have no M protein in their urine, no lytic bone lesions, no anemia, no hypercalcemia, and no renal disease
Smoldering multiple myeloma
M protein level is > 3 g/dL
There is >10% plasma cell involvement on bone marrow biopsy
Affected patients have no lytic bone lesions, no anemia, no hypercalcemia, and no renal disease
Plasma cell leukemia
Peripheral blood contains > 20% plasma cells
M protein levels are low
Affected patients have few bone lesions and few hematologic disturbances
This monoclonal gammopathy occurs in younger patients
Solitary plasmacytoma
Affected patients have only one tumor, with no other bone lesions, and no urine or serum abnormalities
Waldenstrom's macroglobulinemia
IgM M protein is present
Affected patients have hyper viscosity and hypercellular bone marrow with extensive infiltration by lymphoplasma cells
Heavy chain disease
The M protein has an incomplete heavy chain and no light chain
Most of these patients have a large amount of Bence Jones protein (monoclonal free κ or λ chain) in their urine. Thus, the size of the M protein spike is not helpful in excluding MM. If MM still is considered clinically in a patient who does not have an M protein spike on serum protein electrophoresis, urine protein electrophoresis should be performed.44
 
EVALUATION OF AN ABNORMAL SERUM PROTEIN ELECTROPHORESIS
Monoclonal gammopathy is present in up to 8% of healthy geriatric patients. All patients with monoclonal gammopathy require further evaluation to determine the cause of the abnormality. Patients with MGUS require close follow up because about 1% per year develop MM or another malignant monoclonal gammopathy. An algorithm for the follow-up of patients with a monoclonal gammopathy is provided in Figure 1.
If the serum M protein spike is 1.5–2.5 g/dL, it is important to perform nephelometry to quantify the immunoglobulins present and to obtain a 24 hours urine collection for electrophoresis and immunofixation. If these examinations are normal, serum protein electrophoresis should be repeated in 3–6 months; if that examination is normal, serum protein electrophoresis should be repeated annually.
Fig. 1: An algorithm for the follow-up of patients with a monoclonal gammopathy
45
If the repeat examination is abnormal or future patterns are abnormal, the next step is to refer the patient to a hematologist oncologist.
An M protein spike of greater than 2.5 g/dL should be assessed with a metastatic bone survey. In addition, a β2 microglobulin test, a CRP test, and a 24 hours urine collection for electrophoresis and immunofixation should be performed.
If WM or other lymphoproliferative process is suspected, an abdominal computed tomographic scan, and bone marrow aspiration and biopsy should be performed. Abnormalities in any of these tests should result in a referral to a hematologist oncologist. If serum protein electrophoresis is abnormal at any time during the follow up, a referral should be made.
 
HYPERGLOBULINEMIA
Increases in total globulins can result from increases in any or all of the fractions as determined by electrophoresis may indicate:
  • Multiple myeloma
  • Chronic inflammatory disease (e.g. rheumatoid arthritis, systemic lupus erythematosus)
  • Hyperimmunization
  • Acute infection
  • Waldenstrom's macroglobulinemia
  • Chronic liver disease.
 
Alpha Globulins
 
Acute Phase Reactant Response
This usually results in increased α (especially α2) globulins. Acute phase reactants are a diverse group of proteins that increase in serum very rapidly (within 12–24 hours) following tissue injury of any cause (inflammation, acute bacterial and viral infections, necrosis, neoplasia and trauma). Raised serum levels are the result of increased hepatic synthesis mediated by cytokines [interleukin (IL) 1, IL 6 and tumor necrosis factor (TNF)α]. They also tend to remain elevated in chronic inflammatory conditions.
 
Nephrotic Syndrome
A dramatic increase in α2 globulins is often seen (due to very low density lipoprotein and α2 macroglobulin)
  • Drugs: Corticosteroids in dogs will cause a sharp almost monoclonal like peak in the α2 region due to increase in haptoglobin.
Increased α1 globulin proteins may be due to:
  • Acute inflammatory disease
  • Cancer
  • Chronic inflammatory disease [e.g. rheumatoid arthritis, systemic lupus erythematosus (SLE), etc.]
46
Decreased α1 globulin proteins may be a sign of:
  • α1 antitrypsin deficiency
Increased α2 globulin proteins may indicate:
  • Acute inflammation
  • Chronic inflammation
Decreased α2 globulin proteins may indicate:
  • Hemolysis
Increased β globulin proteins may indicate:
  • Hyperlipoproteinemia (e.g. familial hypercholesterolemia)
  • Estrogen therapy
  • β globulins
  • Inflammation (acute and chronic): Increased β globulin often accompanies increases in γ globulins (response to antigenic stimulation)
  • Active liver disease and supportive dermatopathies (both of which are associated with elevated IgM)
  • Nephrotic syndrome (associated with an increase in transferrin and lipoproteins).
Decreased β globulin proteins may indicate:
  • Congenital coagulation disorder
  • Consumptive coagulopathy
  • Disseminated intravascular coagulation
  • Gamma globulins.
Increases in this fraction occur most commonly in conditions in which there is an active immune response to antigenic stimulation usually resulting in a polyclonal gammopathy. Neoplasms of immunoglobulin producing cells (plasma cells, B lymphocytes) can also be responsible for monoclonal increases in this fraction.
 
NEOPLASTIC DISORDERS
 
Multiple Myeloma
Multiple myeloma (from Greek myelo meaning bone marrow), also known as plasma cell myeloma or Kahler's disease (after Otto Kahler), is a cancer of plasma cells, a type of white blood cell s (WBCs) normally responsible for producing antibodies, the most common cause (producing an IgG or IgA monoclonal). Other neoplastic disorders associated with a monoclonal gammopathy include lymphoma (IgM or IgG) and chronic lymphocytic leukemia (usually IgG).
In MM, collection of abnormal plasma cells accumulate in the bone marrow (Fig. 2) where they interfere with the production of normal blood cells. Most cases of myeloma also feature the production of a paraprotein, an abnormal antibody, which can cause kidney problems. Bone lesions and hypercalcemia (high calcium levels) are also often encountered. 47
Fig. 2: Abnormal plasma cells accumulate in the bone marrow in multiple myeloma
Myeloma is diagnosed with blood tests (serum protein electrophoresis, serum free κ/λ light chain assay), bone marrow examination, urine protein electrophoresis, and X-rays of commonly involved bones. Myeloma is generally thought to be treatable but incurable. Remissions may be induced with steroids, chemotherapy, proteasome inhibitors (e.g. bortezomib), immunomodulatory drugs, such as thalidomide or lenalidomide and stem cell transplants. Radiation therapy is sometimes used to reduce pain from bone lesions.
 
Signs and Symptoms
Since many organs can be affected by myeloma, the symptoms and signs vary greatly. A mnemonic sometimes used to remember the common tetrad (four parts) of MM is CRAB: C = Calcium (elevated), R = Renal failure, A = Anemia, B = Bone lesions. Myeloma has many possible symptoms, and all symptoms may be due to other causes. They are presented below in decreasing order of incidence.
Bone pain: Myeloma bone pain usually involves the spine and ribs, and worsens with activity. Persistent localized pain may indicate a pathological bone fracture. Involvement of the vertebrae may lead to spinal cord compression. Myeloma bone disease is due to the overexpression of receptor activator for nuclear factor κB ligand (RANKL) by bone marrow stroma. RANKL activates osteoclasts, which resorb bone. The resultant bone lesions are lytic (cause breakdown) in nature and are best seen in plain radiographs (Fig. 3), which may show “punched out” resorptive lesions (including the “pepper pot” appearance of the skull on radiography). The breakdown of bone also leads to release of calcium into the blood, leading to hypercalcemia and its associated symptoms.
Myeloma bone disease: The most visible aspect of myeloma disease is its effect on bones throughout the body. In the majority of patients with MM, soft spots develop where the bone structure has been damaged. These can extend from the inner bone marrow to the outside surface of the bone. Soft spots appear as “holes” on a standard bone X-ray and are referred to as osteolytic lesions. These lesions weaken the bone, causing pain and increasing the risk of fractures.
Myeloma cells in the bone marrow cause osteolytic lesions, which appear as “holes” on an X-ray. Weakened bones increase the likelihood of fracture. 48
Fig. 3: Multiple myeloma bone lesions
Although it affects the bone, myeloma is considered a hematologic cancer (or blood cancer), because it develops in the B cells of the blood. Treatment of myeloma differs from that of bone cancers (known as sarcomas of the bone).
 
Causes of Bone Destruction in Myeloma
Bone destruction by osteolytic lesions is caused by two separate events. Rapid growth of myeloma cells inhibits normal bone forming cells, damaging bone. In addition, production of substances that activate the cells that resorb bone called osteoclasts is increased. Osteoclasts normally break down old or worn out bone and work with bone forming cells to repair bone. Increased activity of osteoclasts, however, causes bone loss with concomitant loss of bone repair and growth from the suppression of bone formation.
Normal bone cell activity: Normally, osteoclasts function with bone forming cells called osteoblasts to rebuild areas of bone, which are wearing out (fatigued). This process is called bone remodeling, and healthy bone is continually being remodeled.
During the normal process of bone remodeling, the following steps occur:
  1. Osteoclasts are attracted to the area of fatigued bone.
  2. Osteoclasts remove the fatigued bone by breaking it down, creating a cavity in the bone.
  3. Osteoblasts are attracted to the cavity in the bone.
  4. Osteoblasts fill in the cavity with a matrix or framework. Eventually, new bone forms.
Bone cell activity in myeloma: Normally, the activity of the osteoclasts and osteoblasts is well balanced—the osteoclasts clear out the fatigued bone and the osteoblasts begin the rebuilding of new bone. In patients with MM, bone resorption by the osteoclasts is increased and exceeds bone reformation (Fig. 4). Calcium lost from the bones appears in increasing amounts in the 49patient's serum and urine.
Fig. 4: Direct interactions between myeloma cells and bone marrow stromal cells
This increase in bone resorption may result in pain, bone fractures, spinal cord compression, and hypercalcemia.
In myeloma, there is an increase in osteoclast activity that is caused by factors called osteoclastic activating factors (OAFs). These OAFs are known to be released by tumor cells and include a variety of soluble factors known as cytokines.
 
Bone Marrow Microenvironment and its Role in Bone Resorption
The bone marrow microenvironment is the area within the bone (the marrow) where stem cells develop into blood cells and the cells of the immune system. In MM, the bone marrow microenvironment is the area where the malignant plasma cells develop and grow. An important and promising area of myeloma research is the investigation of ways to make the bone marrow microenvironment less hospitable to myeloma cells.
The bone marrow microenvironment plays an important role in the increased bone resorption, which occurs in myeloma. The following steps outline what happens:
  1. Within the bone marrow microenvironment, tumor cells adhere to the bone marrow stromal cells (BMSCs) that are the structural cells of the bone marrow.
  2. Adherence of the MM cells to stromal cells increases the stromal cell production of the growth factor, IL-6 that appears to be necessary for the continued growth and survival of the myeloma cells. Adherence of the myeloma cells to stromal cells also allows the myeloma tumor cells to produce other osteoclast activating factors, including IL 1β and TNF α.
  3. These osteoclast-stimulating factors prompt the bone marrow stromal cells and the osteoblasts to produce yet another growth factor called RANKL.50
  4. Tumor necrosis factor induces the development and growth of osteoclast cells and thus increases osteoclast activity, which results in the bone disease of myeloma.
  5. This increase in osteoclastic activity also results in the release of certain cytokines such as IL-6 that contribute to tumor cell growth and survival.
  6. A clearer understanding of these mechanisms may make it possible to develop more effective treatments to interrupt, slow down, or halt the series of steps, which lead to bone disease and contribute to tumor cell growth and survival in myeloma.
 
Infection
The most common infections are pneumonias and pyelonephritis. Common pneumonia pathogens include Streptococcus pneumoniae, S. aureus and Klebsiella pneumoniae, while common pathogens causing pyelonephritis include Escherichia coli and other gram-negative organisms. The greatest risk period for the occurrence of infection is in the initial few months after the start of chemotherapy. The increased risk of infection is due to immune deficiency. Although the total immunoglobulin level is typically elevated in MM, the majority of the antibodies are ineffective monoclonal antibodies from the clonal plasma cell. A selected group of patients with documented hypogammaglobulinemia may benefit from replacement immunoglobulin therapy to reduce the risk of infection.
 
Renal Failure
Renal failure may develop both acutely and chronically. It is commonly due to hypercalcemia, and/or it may also be due to tubular damage from excretion of light chains (Bence Jones proteins), which can manifest as the Fanconi syndrome (type II renal tubular acidosis). Other causes include glomerular deposition of amyloid, hyperuricemia, recurrent infections (pyelonephritis), and local infiltration of tumor cells.
 
Anemia
The a nemia found in myeloma is usually normocytic and normochromic. It results from the replacement of normal bone marrow by infiltrating tumor cells and inhibition of normal red blood cell production (hematopoiesis) by cytokines.
 
Neurological Symptoms
Common problems are weakness, confusion and fatigue due to hypercalcemia. Headache, visual changes and retinopathy may be the result of hyperviscosity of the blood depending on the properties of the paraprotein. Finally, there may be radicular pain, loss of bowel or bladder control (due to involvement of spinal cord leading to cord compression) or carpal tunnel syndrome and other neuropathies (due to infiltration of peripheral nerves by amyloid). It may give rise to paraplegia in late presenting cases.51
 
Diagnosis
 
Investigations
The presence of unexplained anemia, kidney dysfunction, a high erythrocyte sedimentation rate (ESR), lytic bone lesions, elevated β2 microglobulin and/or a high serum protein, especially raised globulins or immunoglobulin, (the globulin level may be normal in established disease). May show the presence of a paraprotein (M protein) band, with or without reduction of the other (normal) immunoglobulins (known as immune paresis). One type of paraprotein is the Bence Jones protein that is a urinary paraprotein composed of free light chains. Quantitative measurements of the paraproteins are necessary to establish a diagnosis and to monitor the disease. The paraprotein is an abnormal immunoglobulin produced by the tumor clone. Very rarely, the myeloma is nonsecretory (not producing immunoglobulins).
In theory, MM can produce all classes of immunoglobulin, but IgG paraproteins are most common, followed by IgA and IgM. Immunoglobulin D and IgE myelomas are very rare. In addition, light and/or heavy chains (the building blocks of antibodies) may be secreted in isolation—κ or λ light chains or any of the five types of heavy chains (α, γ, δ, ε, or μ heavy chains).
Additional findings include a raised calcium (when osteoclasts are breaking down bone, releasing calcium into the bloodstream) and raised serum creatinine due to reduced renal function, which is mainly due to casts of paraprotein deposition in the kidney, although the cast may also contain complete immunoglobulins, Tamm Horsfall protein and albumin.
 
Workup
The workup of suspected MM includes a skeletal survey. This is a series of X-rays of the skull, axial skeleton, and proximal long bones. Myeloma activity sometimes appear as “lytic lesions” (with local disappearance of normal bone due to resorption) and on the skull X-ray as “punched out lesions” (pepper pot skull). Magnetic resonance imaging (MRI) is more sensitive than simple X-ray in the detection of lytic lesions and may supersede skeletal survey, especially when vertebral disease is suspected. Occasionally, a computed tomography (CT) scan is performed to measure the size of soft tissue plasmacytomas. Bone scans are typically not of any additional value in the workup of myeloma patients (no new bone formation, lytic lesions not well visualized on bone scan).
A bone marrow biopsy is usually performed to estimate the percentage of bone marrow occupied by plasma cells. This percentage is used in the diagnostic criteria for myeloma. Immunohistochemistry (staining particular cell types using antibodies against surface proteins) can detect plasma cells, which express immunoglobulin in the cytoplasm and occasionally on the cell surface. Myeloma cells are typically CD56, CD38, CD138 positive and CD19 and CD45 negative. Cytogenetics may also be performed in myeloma for prognostic purposes, including a myeloma specific fluorescence in situ hybridization (FISH) and virtual karyotype.52
Other useful laboratory tests include quantitative measurement of IgA, IgG, IgM to look for immune paresis, and β2 microglobulin, which provides prognostic information. On peripheral blood smear, the Rouleaux formation of red blood cells is commonly seen, though this is not specific.
The recent introduction of a commercial immunoassay for measurement of free light chains potentially offers an improvement in monitoring disease progression and response to treatment, particularly where the paraprotein is difficult to measure accurately by electrophoresis (for example, in light chain myeloma, or where the paraprotein level is very low). Initial research also suggests that measurement of free light chains may also be used in conjunction with other markers, for assessment of the risk of progression from MGUS to MM.
This assay, the serum free light chain assay, has recently been recommended by the International Myeloma Working Group for the screening, diagnosis, prognosis, and monitoring of plasma cell dyscrasias.
 
Symptomatic Myeloma
  • Clonal plasma cells more than 10% on bone marrow biopsy or (in any quantity) in a biopsy from other tissues (plasmacytoma)
  • An M protein (paraprotein) in either serum or urine (except in cases of true nonsecretory myeloma)
  • Evidence of end organ damage felt related to the plasma cell disorder (related organ or tissue impairment, ROTI, commonly referred to by the acronym “CRAB”)
    • Hypercalcemia (corrected calcium > 2.75 mmol/L)
    • Renal insufficiency attributable to myeloma
    • Anemia (hemoglobin < 10 g/dL)
    • Bone lesions (lytic lesions or osteoporosis with compression fractures).
Note: A recurrent infection alone in a patient who has none of the CRAB features is not sufficient to make the diagnosis of myeloma. Patients who lack CRAB features but have evidence of amyloidosis should be considered as amyloidosis and not myeloma. CRAB like abnormalities are common with numerous diseases, and it is imperative that these abnormalities are felt to be directly attributable to the related plasma cell disorder and every attempt made to rule out other underlying causes of anemia, renal failure, etc.
 
Asymptomatic (Smoldering) Myeloma
  • Serum paraprotein more than 30 g/L
  • Clonal plasma cells more than 10% on bone marrow biopsy
  • No myeloma related organ or tissue impairment.
 
Monoclonal Gammopathy of Undetermined Significance
  • Serum paraprotein less than 30 g/L
  • Clonal plasma cells less than10% on bone marrow biopsy
  • No myeloma related organ or tissue impairment.53
Related conditions include solitary plasmacytoma (a single tumor of plasma cells, typically treated with irradiation), plasma cell dyscrasia (where only the antibodies produce symptoms, e.g. AL amyloidosis), and peripheral neuropathy, organomegaly, endocrinopathy, monoclonal plasma cell disorder, skin changes syndrome (POEMS syndrome).
 
International Staging System
The International Staging System (ISS) for myeloma was published by the International Myeloma Working Group in 2005:
  • Stage I: β2 microglobulin (β2M) less than 3.5 mg/L, albumin greater than equal to 3.5 g/dL
  • Stage II: β2M less than 3.5 mg/L and albumin less than 3.5 g/dL; or β2M 3.5–5.5 mg/L irrespective of the serum albumin
  • Stage III: β2M greater than equal to 5.5 mg/L.
Note that the ISS should be used only in patients who meet diagnostic criteria for myeloma. Patients with MGUS and asymptomatic myeloma who have renal dysfunction from unrelated causes such as diabetes or hypertension may have elevated β2M levels just from the renal dysfunction and cannot be considered as stage III myeloma. This is one of the main drawbacks of the ISS. It does not really quantify tumor burden or extent unlike staging systems used in other cancers. It is more of a prognostic index rather than a true staging system. For this reason, it is recommended that the ISS be used along with the Durie Salmon staging system, which was first published in 1975. The Durie Salmon staging system is still in use.
  • Stage I: all of the following:
    • Hb more than 10 g/dL
    • Normal calcium
    • Skeletal survey: normal or single plasmacytoma or osteoporosis
    • Serum paraprotein level less than 5 g/dL if IgG, less than 3 g/dL if IgA.
    • Urinary light chain excretion less than 4 g/24h.
  • Stage II: fulfills the criteria of neither stage I nor stage III
  • Stage III: one or more of the following:
    • Hb less than 8.5 g/dL
    • High calcium, more than 12 mg/dL
    • Skeletal survey: three or more lytic bone lesions
    • Serum paraprotein more than 7 g/dL if IgG, more than 5 g/dL if IgA
    • Urinary light chain excretion more than 12 g/24 hours
Stages I, II, and III of the Durie Salmon staging system can be divided into A or B depending on serum creatinine levels:
  • A: serum creatinine less than 2 mg/dL (<177 μmol/L)
  • B: serum creatinine more than 2 mg/dL (>177 μmol/L)
 
Pathophysiology
B lymphocytes start in the bone marrow and move to the lymph nodes. As they progress, they mature and display different proteins on their cell surface. When they are activated to secrete antibodies, they are known as plasma cells.54
Multiple myeloma develops in B lymphocytes after they have left the part of the lymph node known as the germinal center. The normal cell line most closely associated with MM cells, is generally taken to be an activated memory B cell or the precursor to plasma cells, the plasmablast.
The immune system keeps the proliferation of B cells and the secretion of antibodies under tight control. When chromosomes and genes are damaged, often through rearrangement, this control is lost. Often, a promoter gene moves (or translocates) to a chromosome where it stimulates an antibody gene to overproduction.
A chromosomal translocation between the immunoglobulin heavy chain gene (on chromosome 14, locus q32) and an oncogene (often 11q13, 4p16.3, 6p21, 16q23 and 20q11) is frequently observed in patients with MM. This mutation results in dysregulation of the oncogene, which is thought to be an important initiating event in the pathogenesis of myeloma. The result is proliferation of a plasma cell clone and genomic instability that leads to further mutations and translocations. The chromosome 14 abnormality is observed in about 50% of all cases of myeloma. Deletion of (parts of) chromosome 13 is also observed in about 50% of cases.
Production of cytokines (especially IL-6) by the plasma cells causes much of their localized damage, such as osteoporosis and creates a microenvironment in which the malignant cells thrive. Angiogenesis (the attraction of new blood vessels) is increased.
The produced antibodies are deposited in various organs, leading to renal failure, polyneuropathy, and various other myeloma associated symptoms.
 
Treatment
Treatment for MM is focused on therapies, which decrease the clonal plasma cell population and consequently decrease the signs and symptoms of disease. If the disease is completely asymptomatic (i.e. there is a paraprotein and an abnormal bone marrow population but no end organ damage), as in smoldering myeloma, treatment is typically deferred.
In addition to direct treatment of the plasma cell proliferation, bisphosphonates (e.g. pamidronate or zoledronic acid) are routinely administered to prevent fractures. They have also been observed to have direct antitumor effect even in patients without known skeletal disease. If needed, red blood cell transfusions or erythropoietin can be used for management of anemia.
 
Initial Therapy
Initial treatment of MM depends on the patient's age and comorbidities. In recent years, high dose chemotherapy with autologous hematopoietic stem cell transplantation has become the preferred choice of treatment for patients under the age of 65. Prior to stem cell transplantation, these patients receive an initial course of induction chemotherapy. The most common induction regimens used today are thalidomide - dexamethasone, bortezomib -based regimens, and lenalidomide -dexamethasone. Autologous stem cell 55transplantation (ASCT), the transplantation of a patient's own stem cells after chemotherapy, is the most common type of stem cell transplantation for MM. It is not curative but does prolong overall survival and complete remission. Allogeneic stem cell transplantation, the transplantation of a healthy person's stem cells into the affected patient, has the potential for a cure but is only available to a small percentage of patients. Furthermore, there is a 5–10% treatment associated mortality rate.
Patients over age 65 and patients with significant concurrent illness often cannot tolerate stem cell transplantation. For these patients, the standard of care has been chemotherapy with melphalan and prednisone. Recent studies among this population suggest improved outcomes with new chemotherapy regimens. Treatment with bortezomib, melphalan, and prednisone had an estimated overall survival of 83% at 30 months; lenalidomide plus low dose dexamethasone had an 82% survival at 2 years and melphalan, prednisone and lenalidomide had a 90% survival at 2 years. Head-to-head studies comparing these regimens have not been performed.
A 2009 review noted, “deep venous thrombosis and pulmonary embolism are the major side effects of thalidomide and lenalidomide. Lenalidomide causes more myelosuppression, while thalidomide causes more sedation. Peripheral neuropathy and thrombocytopenia are major side effects of bortezomib”.
Treatment of related hyperviscosity syndrome may be required to prevent neurologic symptoms or renal failure.
 
Maintenance Therapy
Sometimes after the initial treatment an ongoing maintenance therapy is offered. A 2009 review of maintenance therapy concluded, “In younger patients, post ASCT maintenance therapy with thalidomide appears to increase tumor burden reduction further, which translates in [to] prolonged Progression free survival (PFS).”
A different 2009 review stated, “the role of maintenance therapy with thalidomide, lenalidomide, or bortezomib for patients with multiple myeloma is not definitively established; instead, such therapy should be performed only in the context of a clinical trial”.
 
Relapse
The natural history of myeloma is of relapse following treatment. Depending on the patient's condition, the prior treatment modalities used and the duration of remission, options for relapsed disease include retreatment with the original agent, use of other agents (such as melphalan, cyclophosphamide, thalidomide or dexamethasone, alone or in combination), and a second autologous stem cell transplant.
Later in the course of the disease, “treatment resistance” occurs. This may be a reversible effect, and some new treatment modalities may resensitize the tumor to standard therapy. For patients with relapsed disease, bortezomib is 56a recent addition to the therapeutic arsenal, especially as second line therapy, since 2005. Bortezomib is a proteasome inhibitor. Finally, lenalidomide, a less toxic thalidomide analog, is showing promise for treating myeloma. More and more patients survive longer and longer, thanks to stem cell transplant (with their own or a donor's) and treatments combining Bortezomib, dexamethasone, and melphalan or cyclophosphamide. This seems to maintain the monoclonal peak at a reasonable level. Survival expectancy is then rising, and new treatments are being developed.
Renal failure in MM can be acute (reversible) or chronic (irreversible). Acute renal failure typically resolves when the calcium and paraprotein levels are brought under control. Treatment of chronic renal failure is dependent on the type of renal failure and may involve dialysis.
 
Prognosis
With high-dose therapy followed by autologous stem cell transplantation, the median survival has been estimated in 2003 to be approximately 4.5 years, compared to a median of approximately 3.5 years with “standard” therapy.
The International Staging System can help predict survival with a median survival (in 2005) of 62 months for stage I disease, 45 months for stage II disease, and 29 months for stage III disease.
The prognoses for patients with MM, as those with other diseases, are not the same for everyone. The average age of onset is 70 years. Older patients often are experiencing other serious diseases, which affect survival. Younger patients might have much longer survival rates.
Some myeloma centers now employ genetic testing, which they call a “gene array”. By examining DNA, oncologists can determine, whether patients are at a high risk or low risk of the cancer returning quickly following treatment.
Cytogenetic analysis of myeloma cells may be of prognostic value, with deletion of chromosome 13, nonhyperdiploidy, and the balanced translocations t(4;14) and t(14;16) conferring a poorer prognosis. The 11q13 and 6p21 cytogenetic abnormalities are associated with a better prognosis.
Prognostic markers, such as these are always generated by retrospective analyses, and it is likely that new treatment developments will improve the outlook for those with traditionally “poor risk” disease.
Single nucleotide polymorphism (SNP) array karyotyping can detect copy number alterations of prognostic significance that may be missed by a targeted FISH panel. In MM, lack of a proliferative clone makes conventional cytogenetics informative in only approximately 30% of cases.
Virtual karyotyping identified chromosomal abnormalities in 98% of MM cases.
  • del( 12p13.31) is an independent adverse marker
  • amp(5q31.1) is a favorable marker.
The prognostic impact of amp(5q31.1) overrides that of hyperdiploidy and also identifies patients, who greatly benefit from high dose therapy.57
Array-based karyotyping cannot detect balanced translocations, such as t (4; 14) seen in approximately 15% of MM. Therefore, FISH for this translocation should also be performed if using SNP arrays to detect genome wide copy number alterations of prognostic significance in MM.
Some MM cell lines overproduce TGFβ that inhibits T cell activity. Bacteremia is a common complication of MM, particularly among individuals with low IgM and IgA levels. Osteomyelitis has been reported in three individuals with MM.
Multiple myeloma is the second most prevalent blood cancer (10%) after non-Hodgkin's lymphoma. It represents approximately 1% of all cancers and 2% of all cancer deaths. Although the peak age of onset of MM is 65–70 years of age, recent statistics indicate both increasing incidence and earlier age of onset.
Multiple myeloma affects slightly more men than women. African Americans and Native Pacific Islanders have the highest reported incidence of this disease in the United States and Asians, the lowest. Results of a recent study found the incidence of myeloma to be 9.5 cases per 100,000 African-Americans and 4.1 cases per 100,000 Caucasian-Americans. Among African-Americans, myeloma is one of the top 10 leading causes of cancer death.
 
Waldenström's Macroglobulinemia
Waldenström's macroglobulinemia (WM) is a neoplasm of B cells (lymphoma) that has a different presentation from MM. The main attributing antibody is immunoglobulin M (IgM). Waldenström's macroglobulinemia is an “indolent lymphoma”, (i.e. one that tends to grow and spread slowly). It is a type of lymphoproliferative disease, which shares clinical characteristics with the indolent non-Hodgkin's lymphomas.
As with other lymphomas, the disease is characterized by an uncontrolled increase of B cells, i.e. white blood cells formed in the bone marrow and lymph nodes. The proliferation of B cells interferes with the production of red blood cells, resulting in anemia. A unique characteristic of the disease is that the B cells produce excess amounts of immunoglobulin protein (IgM), thickening the blood, and requiring additional treatment. While the disease is incurable, it is treatable. Because of its indolent nature, many patients are able to lead active lives and when treatment is required, may experience years of symptom free remission.
 
History and Classification
Waldenström's macroglobulinemia was first described by Jan G Waldenström (1906–1996) in 1944 in two patients with bleeding from the nose and mouth, anemia, decreased levels of fibrinogen in the blood (hypofibrinogenemia), swollen lymph nodes, neoplastic plasma cells in bone marrow, and increased viscosity of the blood due to increased levels of a class of heavy proteins called macroglobulins.
For a time, WM was considered to be related to MM due to the presence of monoclonal gammopathy and infiltration of the bone marrow 58and other organs by plasmacytoid lymphocytes. The new World Health Organization (WHO) classification, however, places WM under the category of lymphoplasmacytic lymphomas, itself a subcategory of the indolent (low grade) non-Hodgkin's lymphomas. In recent years, there have been significant advances in the biology and treatment of WM.
 
Causes
Waldenstrom's macroglobulinemia is characterized by an uncontrolled clonal proliferation of terminally differentiated B lymphocytes. The underlying etiology is not yet known, but a number of risk factors have been identified. There has been an association demonstrated with the locus 6p21.3 on chromosome 6. There is a two to three-fold risk increase of developing WM in people with a personal history of autoimmune diseases with autoantibodies and particularly elevated risks associated with hepatitis, human immunodeficiency virus, and rickettsiosis.
There are genetic factors, with first degree relatives shown to have a highly increased risk of also contracting Waldenström's. There is also evidence to suggest that environmental factors, including exposure to farming, pesticides, wood dust, and organic solvents may influence the development of Waldenstrom's.
 
Biochemistry
Although believed to be a sporadic disease, studies have shown increased susceptibility within families, indicating a genetic component. However, genetic involvement is poorly understood. WM cells show only minimal changes in cytogenetic and gene expression studies. Comparative genomic hybridization identified the following chromosomal abnormalities : deletions of 6q23 and 13q14 and gains of 3q13-q28, 6p and 18q. FGFR3 is overexpressed. The following signaling pathways have been implicated:
  • CD154 / CD40
  • Akt
  • ubiquitination, p53 activation, cytochrome C release
  • NF κB
  • WNT / β catenin
  • mTOR
  • ERK
  • MAPK
  • Bcl 2.
The protein Src tyrosine kinase is overexpressed in WM cells compared with control B cells. Inhibition of Src arrests the cell cycle at phase G1 and has little effect on the survival of WM or normal cells.
MicroRNAs involved in Waldenstrom's:
  • Increased expression of miRNAs-363, -206, -494, -155, -184, -542–3p
  • Decreased expression of miRNA-9
  • MicroRNA-155 regulates the proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT and NF κB pathways.
59
In WM cells, histone deacetylases and histone modifying genes are deregulated.
Bone marrow tumor cells express the following antigen targets: CD20 (98.3%), CD22 (88.3%), CD40 (83.3%), CD52 (77.4%), IgM (83.3%), MUC1 core protein (57.8%), and 1D10 (50%).
 
Epidemiology
Of all cancers involving the same class of blood cell, 1% of cases are WM. It is a rare disorder, with fewer than 1,500 cases occurring in the United States annually. The median age of onset of WM is between 60 and 65 years, with some cases occurring in late teens.
 
Symptoms
Symptoms of WM include weakness, fatigue, weight loss, and chronic oozing of blood from the nose and gums. Peripheral neuropathy can occur in 10% of patients. Lymphadenopathy, splenomegaly, and/or hepatomegaly are present in 30–40% of cases. Other possible symptoms include blurring or loss of vision, headache, and (rarely) stroke or coma.
 
Pathophysiology
Symptoms blurring or loss of vision, headache, and (rarely) stroke or coma are due to the effects of the IgM paraprotein that may cause autoimmune phenomenon or cryoglobulinemia. Other symptoms of WM are due to the hyperviscosity syndrome, which is present in 6–20% of patients. This is attributed to the IgM M protein increasing the viscosity of the blood by forming aggregates to each other, binding water through their carbohydrate component, and by their interaction with blood cells.
 
Diagnosis
A diagnosis of WM depends on a significant monoclonal IgM spike evident in blood tests and malignant cells consistent with the disease in bone marrow biopsy samples. Blood tests show the level of IgM in the blood and the presence of proteins or tumor markers, which are the key symptoms of WM. A bone marrow biopsy provides a sample of bone marrow, usually from the back of the pelvis bone. The sample is extracted through a needle and examined under a microscope. A pathologist identifies the particular lymphocytes that indicate WM. Flow cytometery may be used to examine markers on the cell surface or inside the lymphocytes.
Additional tests such as CT scan may be used to evaluate the chest, abdomen, and pelvis, particularly swelling of the lymph nodes, liver and spleen. A skeletal survey can help distinguish between WM and MM. Anemia is typically found in 80% of patients with WM. Leukopenia and thrombocytopenia may be observed. Neutropenia may also be found in some patients.60
Laboratory tests include lactate dehydrogenase (LDH) levels, uric acid levels, ESR, renal and hepatic functions, total protein levels, and an albumin to globulin ratio. The ESR and uric acid level may be elevated. Creatinine is occasionally elevated and electrolytes are occasionally abnormal. Hypercalcemia is noted in approximately 4% of patients. The LDH level is frequently elevated, indicating the extent of WM-related tissue involvement. Rheumatoid factor, cryoglobulins, direct antiglobulin test, and cold agglutinin titer results can be positive. β2 microglobulin and CRP test results are not specific for WM. β2 microglobulin is elevated in proportion to tumor mass. Coagulation abnormalities may be present. Prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen tests should be performed. Platelet aggregation studies are optional. Serum protein electrophoresis results indicate evidence of a monoclonal spike but cannot establish the spike as IgM. An M component with β to γ mobility is highly suggestive of WM. Immunoelectrophoresis and immunofixation studies help identify the type of immunoglobulin, the clonality of the light chain, and the monoclonality and quantitation of the paraprotein. High resolution electrophoresis, and serum, and urine immunofixation are recommended to help identify and characterize the monoclonal IgM paraprotein.
The light chain of the M protein is usually the κ light chain. At times, patients with WM may exhibit more than one M protein. Plasma viscosity must be measured. Results from characterization studies of urinary immunoglobulins indicate that light chains (Bence Jones protein), usually of the κ type, are found in the urine. Urine collections should be concentrated.
Bence Jones proteinuria is observed in approximately 40% of patients and exceeds 1 g/day in approximately 3% of patients. Patients with findings of peripheral neuropathy should have nerve conduction studies and antimyelin associated glycoprotein serology.
 
Prognosis
Current medical treatments result in survival of some longer than 10 years; in part, this is because better diagnostic testing means early diagnosis and treatments. Older diagnosis and treatments resulted in published reports of median survival of approximately 5 years from time of diagnosis. Currently, median survival is 6.5 years. In rare instances, WM progresses to MM.
 
Treatment
There is no single accepted treatment for WM. There is marked variation in clinical outcome due to gaps in knowledge of the disease's molecular basis. Objective response rates are high (> 80%), but complete response rates are low (0–15%).
Waldenström's macroglobulinemia patients are at higher risk of developing second cancers than the general population; however, it is not yet clear whether treatments are contributory.
Watchful waiting: In the absence of symptoms, many clinicians will recommend simply monitoring the patient. But on occasion, the disease can 61be fatal. In 1991, Waldenström himself raised the question of the need for effective therapy.
First line: Treatment should be started. It should address both the paraprotein level and the lymphocytic B cells.
In 2002, a panel at the International Workshop on Waldenström macroglobulinemia agreed on criteria for the initiation of therapy. They recommended starting therapy in patients with constitutional symptoms, such as recurrent fever, night sweats, fatigue due to anemia, weight loss, progressive symptomatic lymphadenopathy or splenomegaly, and anemia due to marrow infiltration. Complications, such as hyperviscosity syndrome, symptomatic sensorimotor peripheral neuropathy, systemic amyloidosis, renal insufficiency, or symptomatic cryoglobulinemia were also suggested as indications for therapy.
Treatment includes the monoclonal antibody rituximab, sometimes in combination with chemotherapeutic drugs, such as chlorambucil, cyclophosphamide, or vincristine or thalidomide. Corticosteroids, such as p rednisone, may also be used in combination. Plasmapheresis can be used to treat the hyperviscosity syndrome by removing the paraprotein from the blood, although it does not address the underlying disease.
Recently, autologous bone marrow transplantation has been added to the available treatment options.
Salvage therapy: When primary or secondary resistance invariably develops, salvage therapy is considered. Allogeneic stem cell transplantation can induce durable remissions for heavily pretreated patients.
Patient stratification: Patients with polymorphic variants (alleles) FCGR3A -48 and -158 were associated with improved categorical responses to rituximab-based treatments.
 
NON-NEOPLASTIC DISORDERS
Monoclonal gammopathies (usually IgG) have been reported with occult heartworm disease, FIPV (rarely), Ehrlichia canis, lymphoplasmacytic enteritis, lymphoplasmacytic dermatitis, and amyloidosis. These causes should be ruled out before a diagnosis of multiple myeloma is made in a patient with an IgG monoclonal gammopathy. However, these previous reports possibly misinterpreted a “restricted oligoclonal” gammopathy as a true monoclonal gammopathy (see the electrophoresis page for more information) and a true IgG monoclonal gammopathy is likely only seen with B lymphoid (B CLL, B cell lymphoma), or plasma cell neoplasia (extramedullary plasmacytoma or multiple myeloma) rather than these reactive causes.
 
Monoclonal Gammopathy of Unknown Significance
Monoclonal gammopathy of unknown significance or undetermined significance is a common precancerous condition affecting people 50 years of age and older. It is a noncancerous (benign) condition. It was first described by Mayo Clinic researchers in 1978 and is characterized by the presence of an 62abnormal protein in the blood called a (monoclonal) protein or M protein. MGUS has a small risk (1% each year) of progressing to a blood cancer called multiple myeloma or a related condition.
Monoclonal gammopathy of unknown significance or undetermined significance is a condition where the body makes an abnormal protein called a paraprotein that is found in the blood. MGUS is linked to the immune system, which helps the body fight infection and disease. The immune system is made up of organs, such as the bone marrow, the spleen, lymph nodes (or lymph glands), and a type of WBC called lymphocytes.
Lymphocytes start to grow in the bone marrow, i.e. where blood cells are made. The two main types of lymphocytes are B cells and T cells. Some B cell lymphocytes develop into plasma cells and make antibodies to help fight infections. Antibodies are made from a protein called immunoglobulin.
Monoclonal gammopathy of unknown significance or undetermined significance occurs when particular plasma cells produce abnormally large amounts of one type of antibody. This abnormal antibody or immunoglobulin is called a paraprotein (or M protein). The paraprotein does not do anything useful, and it is not harmful. MGUS is not a cancer. Some cancers, such as myeloma (a cancer of the plasma cell) and lymphoma (cancer of the lymphatic system) also produce large amounts of paraproteins. Although the levels of paraprotein are raised in MGUS, but they are not as high as the amount produced in people with cancer. Most people with MGUS remain well and never have any problems related to it, a small number of people may go on to develop more serious problems, so everyone with MGUS has a regular check-up. MGUS is much more common in older people. The cause of MGUS is unknown. It is more common in people with conditions that affect the immune system, such as rheumatoid arthritis and certain infections.
 
Signs and Symptoms
Monoclonal gammopathy of unknown significance or undetermined significance is usually found by chance, often during a blood test carried out for some other reasons. It does not usually cause any symptoms. Occasionally, people with MGUS have numbness or tingling in their hands and feet, or problems with their balance. This may be due to damage to nerves caused by the paraprotein in the blood. If the symptoms get worse, the patient should be referred to a neurologist.
 
Diagnosis
Usually seen by a clinical biochemist and hematologist, who are specialized in blood disorders. Other tests may also advise to be done to rule out more serious conditions, such as myeloma or lymphoma. These tests may include X-rays, scans, and occasionally, a bone marrow test.
 
Blood Tests
Serum protein electrophoresis is done to diagnose and monitor MGUS. This test measures the type and amount of paraprotein produced by the 63plasma cells. Full blood count should be checked out to make sure that bone marrow is working well, since bone marrow is a part of the immune system.
Blood tests may also be taken to check the functioning of liver and kidney, as well as the level of calcium that may be raised in conditions, such as myeloma.
 
Urine Tests
Urine sample should be checked for the paraprotein.
 
X-rays
Some people may have a number of X-rays taken of different bones in the body (called a skeletal survey). This is to rule out any damage to the bones, which may be linked with myeloma.
 
CT Scan
This may be used to check if any lymph nodes or the liver or spleen are enlarged. A CT scan takes a series of X-rays that build up a three dimensional.
 
Treatment and Follow-up
Monoclonal gammopathy of unknown significance or undetermined significance does not need treatment, as it does not cause any symptoms. In most people MGUS remains stable and will never cause any problems. Because of the very small risk of MGUS developing into a cancer, such as myeloma or lymphoma, regular checkups are important. The lower the level of the paraprotein, the less chance there is of this happening. Usually paraprotein levels should be checked every 3–4 months for the first year. If the paraprotein level remains steady and there are no other problems, the interval between appointments will become longer, but in case, if the paraprotein levels are rising, or if you develop symptoms, tests ma y need to be repeated or new tests may be carried out.
Decreased total protein may indicate:
  • Cirrhosis
  • Malnutrition
  • Nephrotic syndrome
  • Gastrointestinal protein losing enteropathy.
 
CLINICAL SIGNIFICANCE OF CEREBROSPINAL FLUID ELECTROPHORESIS
Many disorders of the central nervous system (CNS) are associated with increased concentration of cerebrospinal fluid (CSF) proteins, either due to increase in the permeability of blood CSF barrier or to synthesis of immunoglobulins, primarily IgG, within the CNS. The intrathecal synthesis of Ig is often associated with Ig's heterogeneity, which manifests itself as “oligoclonal banding” seen in high resolution electrophoretic migration 64patterns. The bands in the γ globulin zone are not always the true oligoclonal bands, i.e. IgG, A, or M and, therefore, do not have the same diagnostic significance of the oligoclonal Ig bands. Immunofixation is a choice technique, since it can prove the Ig character of the oligoclonal bands and can identify the Ig involved. To confirm intrathecal Ig synthesis, patient serum and CSF must be analyzed in parallel to demonstrate differences in Ig's distribution patterns between CSF and serum. Confirmation of intrathecal Ig synthesis is important information to suspect inflammatory disease of the CNS, such as caused by multiple sclerosis (MS).
Cerebrospinal fluid studies can confirm demyelinating disease of the nervous system. They show an increase in immunoglobulin concentrations in more than 90% of patients with MS. IgG index (a comparison between IgG levels in the CSF and in the serum) is elevated in many MS patients. Oligoclonal immunoglobulin bands (Fig. 5) can be identified in the CSF of MS patients via electrophoresis. The overall protein level is also slightly elevated up to 0.1 g/L. Protein level can be higher if the patient is going through a marked relapse (i.e. severe optic neuritis). Some patients also exhibit a slight elevation in cell count (up to 50 per cubic millimeter). Most cells can be identified as T lymphocytes.
Cerebrospinal fluid analysis is a set of laboratory tests that examine a sample of the fluid surrounding the brain and spinal cord. This fluid is an ultrafiltrate of plasma. It is clear and colorless. It contains glucose, electrolytes, amino acids, and other small molecules found in plasma but has very little protein and few cells.
Cerebrospinal fluid protects the central nervous system from injury, cushions it from the surrounding bone structure, provides it with nutrients, and removes waste products by returning them to the blood. CSF is withdrawn from the subarachnoid space through a needle by a procedure called a lumbar puncture or spinal tap. CSF analysis includes tests in clinical chemistry, hematology, immunology, and microbiology.
Fig. 5: Oligoclonal immunoglobulin bands in cerebrospinal fluid sample
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Usually three or four tubes are collected. The first tube is used for chemical and/or serological analysis, and the last two tubes are used for hematology and microbiology tests. This reduces the chances of a falsely elevated white cell count caused by a traumatic tap (bleeding into the subarachnoid space at the puncture site) and contamination of the bacterial culture by skin germs or flora.
 
Purpose
The purpose of a CSF analysis is to diagnose medical disorders, which affect the CNS, such as:
  • Multiple sclerosis, a degenerative nerve disease that results in the loss of the myelin coating of the nerve fibers of the brain and spinal cord
  • Meningitis and encephalitis that may be viral, bacterial, fungal, or parasitic infections
  • Metastatic tumors (e.g. leukemia) and central nervous system tumors that shed cells into the CSF
  • Syphilis, a sexually transmitted bacterial disease
  • Bleeding (hemorrhaging) in the brain and spinal cord
  • Guillain–Barré syndrome (GBS), a demyelinating disease involving peripheral sensory and motor nerves
  • Lyme disease
  • Systemic lupus erythematosus
  • Neurosarcoidosis
  • Subacute sclerosing panencephalitis
  • Primary CNS lymphoma
  • Sjogren's syndrome.
Routine examination of CSF includes visual observation of color and clarity and tests for glucose, protein, lactate, lactate dehydrogenase, red blood cell count, white blood cell count with differential, syphilis serology (testing for antibodies indicative of syphilis), Gram stain, and bacterial culture. Further tests may need to be performed depending upon the results of initial tests and the presumptive diagnosis. For example, an abnormally high total protein seen in a patient suspected of having a demyelinating disease, such as MS dictates CSF protein electrophoresis and measurement of immunoglobulin levels and myelin basic protein.
 
Gross Examination
Color and clarity are important diagnostic characteristics of CSF. Straw, pink, yellow, or amber pigments (xanthochromia) are abnormal and indicate the presence of bilirubin, hemoglobin, red blood cells, or increased protein. Turbidity (suspended particles) indicates an increased number of cells. Gross examination is an important aid in differentiating a subarachnoid hemorrhage from a traumatic tap. The latter is often associated with sequential clearing of CSF as it is collected, streaks of blood in an otherwise clear fluid, or a sample that clots.66
 
Glucose
Cerebrospinal fluid glucose is normally approximately two-thirds of the fasting plasma glucose. A glucose level below 40 mg/dL is significant and occurs in bacterial and fungal meningitis and in malignancy.
 
Protein
Total protein levels in CSF are normally very low, and albumin makes up approximately two-thirds of the total. High levels are seen in many conditions including bacterial and fungal meningitis, MS, tumors, subarachnoid hemorrhage, and traumatic tap.
 
Lactate
The CSF lactate is used mainly to help differentiate bacterial and fungal meningitis, which cause increased lactate from viral meningitis, which does not.
 
Lactate Dehydrogenase
This enzyme is elevated in bacterial and fungal meningitis, malignancy, and subarachnoid hemorrhage.
 
White Blood Cell Count
The number of WBCs in CSF is very low, usually necessitating a manual WBC count. An increase in WBCs may occur in many conditions, including infection (viral, bacterial, fungal, and parasitic), allergy, leukemia, MS, hemorrhage, traumatic tap, encephalitis, and GBS. The WBC differential helps distinguish many of these causes. For example, viral infection is usually associated with an increase in lymphocytes, while bacterial and fungal infections are associated with an increase in polymorph nuclear leukocytes (neutrophils). The differential may also reveal eosinophils associated with allergy and ventricular shunts; macrophages with ingested bacteria (indicating meningitis), RBCs (indicating hemorrhage), or lipids (indicating possible cerebral infarction); blasts (immature cells) that indicate leukemia; and malignant cells characteristic of the tissue of origin. About 50% of metastatic cancers, which infiltrate the central nervous system and about 10% of central nervous system tumors will shed cells into the CSF.
 
Red Blood Cell Count
While not normally found in CSF, RBCs will appear whenever bleeding has occurred. Red cells in CSF signal subarachnoid hemorrhage, stroke, or traumatic tap. Since white cells may enter the CSF in response to local infection, inflammation, or bleeding, the RBC count is used to correct the WBC count so that it reflects conditions other than hemorrhage or a traumatic tap. This is accomplished by counting RBCs and WBCs in both blood and CSF. The ratio of RBCs in CSF to blood is multiplied by the blood WBC count. 67This value is subtracted from the CSF WBC count to eliminate WBCs derived from hemorrhage or traumatic tap.
 
Gram Stain
The Gram stain is performed on sediment of the CSF and is positive in at least 60% of cases of bacterial meningitis. Culture is performed for both aerobic and anerobic bacteria. In addition, other stains (e.g. the acid-fast stain for Mycobacterium tuberculosis, fungal culture, and rapid identification tests [tests for bacterial and fungal antigens]) may be performed routinely.
 
Syphilis Serology
This involves testing for antibodies that indicate neurosyphilis. The fluorescent treponemal antibody absorption (FTA-ABS) test is often used and is positive in persons with active and treated syphilis. The test is used in conjunction with the VDRL test for nontreponemal antibodies, which is positive in most persons with active syphilis, but negative in treated cases.
 
Normal Results
Gross appearance: Normal CSF is clear and colorless
Cerebrospinal fluid opening pressure: 50–175 mm H2O
Specific gravity: 1.006–1.009
Glucose: 40–80 mg/dL
Total protein: 15–45 mg/dL
LD: 1/10 of serum level
Lactate: Less than 35 mg/dL
Leukocytes (white blood cells): 0–5/μL (adults and children); up to 30/μL (newborns)
Differential: 60–80% lymphocytes; up to 30% monocytes and macrophages; other cells 2% or less. Monocytes and macrophages are somewhat higher in neonates
Gram stain: Negative.
Culture: Sterile
Syphilis serology: Negative
Red blood cell count: Normally, there are no red blood cells in the CSF unless the needle passes through a blood vessel on route to the CSF.
 
Diseases Associated
The presence of oligoclonal bands in CSF combined with their absence in blood serum often indicates that immunoglobulins are produced in CNS. Therefore, it is normal to subtract bands in serum from bands in CSF when investigating CNS diseases. Oligoclonal bands are an important indicator in the diagnosis of MS.68
Approximately 79–90% of all patients with MS have permanently observable oligoclonal bands. The presence of one band (a monoclonal band) may be considered serious, such as lymphoproliferative disease, or may simply be normal—it must be interpreted in the context of each specific patient; so, more bands may reflect the presence of a disease. The bands tend to disappear from the cerebrospinal fluid as a person recovers from the neurological disease.
 
MULTIPLE SCLEROSIS
Also known as “disseminated sclerosis” or “encephalomyelitis disseminata”, is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms. Disease onset usually occurs in young adults, and it is more common in women. It has a prevalence that ranges between 2 and 150 per 100,000. MS was first described in 1868 by Jean Martin Charcot.
Multiple sclerosis affects the ability of nerve cells in the brain and spinal cord to communicate with each other effectively. Nerve cells communicate by sending electrical signals called action potentials down long fibers called axons that are contained within an insulating substance called myelin. In MS, the body's own immune system attacks and damages the myelin. When myelin is lost, the axons can no longer effectively conduct signals. The name MS refers to scars (scleroses—better known as plaques or lesions), particularly in the white matter of the brain and spinal cord, which is mainly composed of myelin. Although much is known about the mechanisms involved in the disease process, the cause remains unknown. Theories include genetics or infections. Different environmental risk factors have also been found.
Almost any neurological symptom can appear with the disease, and often progresses to physical and cognitive disability. MS takes several forms, with new symptoms occurring either in discrete attacks (relapsing forms) or slowly accumulating over time (progressive forms). Between attacks, symptoms may go away completely, but permanent neurological problems often occur, especially as the disease advances.
Ninety percent of people with MS have been found to have elevated amounts of an antibody protein called immunoglobin G (IgG) in their CSF to a greater degree than is present in their blood serum. A sample is considered positive for CSF oligoclonal bands, if there are two or more bands in the CSF immunoglobulin region that are not present in the serum (see Fig. 5). Capillary electrophoresis will show these increased levels as IgG oligoclonal bands. By applying a simple formula to the capillary electrophoresis results of both fluids, it is possible to determine the CSF IgG index, the CSF IgG synthesis rate, and CSF IgG/albumin ratio.
During MS relapses, elevated levels of myelin basic protein (MBP) are often detectable through electrophoresis as are other proteins. None of the protein bands are definitive for MS, and up to 10% of CSF samples from people with clinically definite MS can yield negative electrophoresis results.69
There is no known cure for MS. Treatments attempt to return function after an attack, prevent new attacks, and prevent disability. MS medications can have adverse effects or be poorly tolerated, and many people pursue alternative treatments, despite the lack of supporting scientific study. The prognosis is difficult to predict; it depends on the subtype of the disease, the individual's disease characteristics, the initial symptoms and the degree of disability the person experiences as time advances. Life expectancy of people with MS is 5–10 years lower than that of the unaffected population.
 
History
The French neurologist, Jean Martin Charcot (1825–1893) was the first person to recognize MS as a distinct disease in 1868. Summarizing previous reports and adding his own clinical and pathological observations, Charcot called the disease sclerose en plaques. The three signs of MS now known as Charcot's triad are nystagmus, intention tremor, and telegraphic speech (scanning speech), though these are not unique to MS. Charcot also observed cognition changes, describing his patients as having a “marked enfeeblement of the memory” and “conceptions that formed slowly”.
Prior to Charcot, Robert Carswell (1793–1857), a British professor of pathology and Jean Cruveilhier (1791–1873), a French professor of pathologic anatomy had described and illustrated many of the disease's clinical details but did not identify it as a separate disease. Specifically, Carswell described the injuries he found as “a remarkable lesion of the spinal cord accompanied with atrophy”. Under the microscope, Swiss pathologist, Georg Eduard Rindfleisch (1836–1908) noted in 1863 that the inflammation associated lesions were distributed around blood vessels.
After Charcot's description, Eugène Devic (1858–1930), Jozsef Balo (1895–1979), Paul Ferdinand Schilder (1886–1940), and Otto Marburg (1874–1948) described special cases of the disease. During all the 20th century, there was an important development on the theories about the cause and pathogenesis of MS, while efficacious treatments began to appear in 1990.
 
Historical Cases
There are several historical accounts of a person who lived before or shortly after the disease was described by Charcot and probably had MS.
A young woman called Halldora who lived in Iceland around 1200 suddenly lost her vision and mobility but, after praying to the saints, recovered those seven days after. Saint Lidwina of Schiedam (1380–1433), a Dutch nun, may be one of the first clearly identifiable MS patients. From the age of 16 until her death at 53, she suffered intermittent pain, weakness of the legs, and vision loss—symptoms typical of MS. Both cases have led to the proposal of a “Viking gene” hypothesis for the dissemination of the disease.
Augustus Frederick d'Este (1794–1848), son of Prince Augustus Frederick, Duke of Sussex and Lady Augusta Murray and the grandson of George III of the United Kingdom, almost certainly suffered from MS. D'Este left a detailed diary describing his 22 years living with the disease. His diary began in 1822 70and ended in 1846, although it remained unknown until 1948. His symptoms began at age 28 with a sudden transient visual loss (amaurosis fugax) after the funeral of a friend. During the course of his disease, he developed weakness of the legs, clumsiness of the hands, numbness, dizziness, bladder disturbances, and erectile dysfunction. In 1844, he began to use a wheelchair.
Another early account of MS was kept by the British diarist WNP Barbellion, nom de plume of Bruce Frederick Cummings (1889–1919), who maintained a detailed log of his diagnosis and struggle with MS.
 
Main Symptoms of Multiple Sclerosis
A person with MS can suffer almost any neurological symptom or sign, including changes in sensation, such as loss of sensitivity or tingling, pricking or numbness (hypoesthesia and paresthesia), muscle weakness, clonus, muscle spasms, or difficulty in moving; difficulties with coordination and balance (ataxia ); problems in speech (dysarthria) or swallowing (dysphagia ); visual problems (nystagmus, optic neuritis), fatigue, acute or chronic pain ; and bladder and bowel difficulties. Cognitive impairment of varying degrees and emotional symptoms of depression or unstable mood are also common Uhthoff's phenomenon, an exacerbation of extant symptoms due to an exposure to higher than usual ambient temperatures, and Lhermitte's sign, an electrical sensation that runs down the back when bending the neck, are particularly characteristic of MS although not specific (Fig. 6). The main clinical measure of disability progression and symptom severity is the expanded disability status scale or EDSS (Fig. 7).
Symptoms of MS usually appear in episodic acute periods of worsening (called relapses, exacerbations, bouts, attacks, or “flare ups”), in a gradually progressive deterioration of neurologic function, or in a combination of both. Multiple sclerosis relapses are often unpredictable, occurring without warning and without obvious inciting factors with a rate rarely above one and a half per year.
Fig. 6: Main symptoms of multiple sclerosis
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Fig. 7: Expanded disability status scale (EDSS)
Some attacks, however, are preceded by common triggers. Relapses occur more frequently during spring and summer. Viral infections, such as the common cold, influenza, or gastroenteritis increase the risk of relapse. Stress may also trigger an attack. Pregnancy affects the susceptibility to relapse, with a lower relapse rate at each trimester of gestation. During the first few months after delivery, however, the risk of relapse is increased. Overall, pregnancy does not seem to influence long-term disability. Many potential triggers have been examined and found not to influence MS relapse rates. There is no evidence that vaccination and breastfeeding, physical trauma, or Uhthoff's phenomenon is relapse triggers.
 
Causes
Most likely MS occurs as a result of some combination of genetic, environmental and infectious factors, and possibly other factors like vascular problems. Epidemiological studies of MS have provided hints on possible causes for the disease. Theories try to combine the known data into plausible explanations, but none has proved definitive.
 
Genetics
Multiple sclerosis is not considered a hereditary disease. However, a number of genetic variations have been shown to increase the risk of developing the disease.
The risk of acquiring MS is higher in relatives of a person with the disease than in the general population, especially in the case of siblings, parents, and children. The disease has an overall familial recurrence rate of 20%. In the case of monozygotic twins, concordance occurs only in about 35% of cases, 72while it goes down to around 5% in the case of siblings and even lower in half siblings. This indicates susceptibility is partly polygenically driven. It seems to be more common in some ethnic groups than others.
Apart from familial studies, specific genes have been linked with MS. Differences in the human leukocyte antigen (HLA) system—a group of genes in chromosome 6 that serves as the major histocompatibility complex (MHC) in humans—increase the probability of suffering MS. The most consistent finding is the association between MS and alleles of the MHC defined as DR15 and DQ6 (Fig. 8).
 
Environmental Factors
Different environmental factors, both of infectious and noninfectious origin, have been proposed as risk factors for MS. Although some are partly modifiable, only further research, especially clinical trials will reveal whether their elimination can help prevent MS.
Multiple sclerosis is more common in people who live farther from the equator, although many exceptions exist. Decreased sunlight exposure has been linked with a higher risk of MS. Decreased vitamin D production and intake has been the main biological mechanism used to explain the higher risk among those less exposed to sun.
Fig. 8: Human leukocyte antigen region of chromosome 6. Changes in this area increase the probability of suffering MS
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Severe stress may be a risk factor, although evidence is weak. Smoking has also been shown to be an independent risk factor for developing MS. Association with occupational exposures and toxins mainly solvents has been evaluated, but no clear conclusions have been reached. Vaccinations were investigated as causal factors for the disease; however, most studies show no association between MS and vaccines. Several other possible risk factors, such as diet and hormone intake, have been investigated; however, evidence on their relation with the disease is “sparse and unpersuasive”.
Gout occurs less than would statistically be expected in people with MS, and low levels of uric acid have been found in people with MS as compared to normal individuals. This led to the theory that uric acid protects against MS, although its exact importance remains unknown.
 
Infections
Many microbes have been proposed as potential infectious triggers of MS, but none have been substantiated. Moving at an early age from one location in the world to another alters a person's subsequent risk of MS. An explanation for this could be that some kind of infection, produced by a widespread microbe rather than a rare pathogen, is the origin of the disease. There are a number of proposed mechanisms, including the hygiene hypothesis and the prevalence hypothesis. The hygiene hypothesis proposes that exposure to several infectious agents early in life is protective against MS, the disease being a response to a later encounter with such agents. The prevalence hypothesis proposes that the disease is due to a pathogen more common in regions of high MS prevalence wherein most individuals it causes an asymptomatic persistent infection. Only in a few cases and after many years does it cause demyelination. The hygiene hypothesis has received more support than the prevalence hypothesis.
Evidence for viruses as a cause includes the presence of oligoclonal bands in the brain and CSF of most people with MS, the association of several viruses with human demyelination encephalomyelitis, and induction of demyelination in animals through viral infection. Human herpes viruses are a candidate group of viruses linked to MS. Individuals who have never been infected by the Epstein Barr virus have a reduced risk of having the disease, and those infected as young adults have a greater risk than those who had it at a younger age. Although some consider that this goes against the hygiene hypothesis, since the noninfected have probably experienced a more hygienic upbringing, others believe that there is no contradiction, since it is a first encounter at a later moment with the causative virus that is the trigger for the disease. Other diseases that have also been related with MS are measles, mumps, and rubella.
 
Autoimmunology
Multiple sclerosis is believed to be an immune-mediated disorder mediated by a complex interaction of the individual's genetics and, as yet, unidentified environmental insults. Damage is believed to be caused by the person's own 74immune system attacking the nervous system, possibly as a result of exposure to a molecule with a similar structure to one of its own.
 
Lesions
Multiple sclerosis lesions most commonly involve white matter areas close to the ventricles of the cerebellum (Fig. 9), brainstem, basal ganglia and spinal cord, and the optic nerve. The function of white matter cells is to carry signals between gray matter areas, where the processing is done, and the rest of the body. The peripheral nervous system is rarely involved.
More specifically, MS destroys oligodendrocytes, the cells responsible for creating and maintaining a fatty layer known as the myelin sheath (Fig. 10), which helps the neurons carry electrical signals (action potentials). Multiple sclerosis results in a thinning or complete loss of myelin and, as the disease advances, the cutting (transection) of the neuron's axons. When the myelin is lost, a neuron can no longer effectively conduct electrical signals. A repair process, called remyelination, takes place in early phases of the disease, but the oligodendrocytes cannot completely rebuild the cell's myelin sheath. Repeated attacks lead to successively lesser effective remyelinations, until a scar like plaque is built up around the damaged axons, different lesion patterns have been described.
 
Inflammation
Apart from demyelination, the other pathologic hallmark of the disease is inflammation. According to a strictly immunological explanation of MS, the inflammatory process is caused by T cells, a kind of lymphocyte. Lymphocytes are cells that play an important role in the body's defenses. In MS, T cells gain entry into the brain via disruptions in the blood-brain barrier. Evidence from animal models also point to a role of B cells in addition to T cells in development of the disease.
The T cells recognize myelin as foreign and attack it, as if it were an invading virus. This triggers inflammatory processes, stimulating other immune cells and soluble factors like cytokines and antibodies.
Figure 9: Visualization of small callosal multiple sclerosis lesions
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Fig. 10: Nerve axon with myelin sheath
Further leaks form in the blood-brain barrier, which in turn, cause a number of other damaging effects such as swelling, activation of macrophages, and more activation of cytokines and other destructive proteins.
 
Blood–brain Barrier Breakdown
The blood–brain barrier is a capillary system that normally prevents entry of T cells into the central nervous system. However, it may become permeable to these types of cells, due to an infection or a virus. When the blood–brain barrier regains its integrity, typically after the infection or virus has cleared, the T cells are trapped inside the brain.
 
Diagnosis
Multiple sclerosis can be difficult to diagnose, since its signs and symptoms may be similar to other medical problems. Medical organizations have created diagnostic criteria to ease and standardize the diagnostic process, especially in the first stages of the disease. Historically, the Schumacher and Poser criteria were both popular.76
Currently, the McDonald criteria focus on a demonstration with clinical, laboratory, and radiologic data of the dissemination of MS lesions in time and space for noninvasive MS diagnosis, though some have stated that the only proved diagnosis of MS is autopsy, or occasionally biopsy, where lesions typical of MS can be detected through histopathological techniques.
Clinical data alone may be sufficient for a diagnosis of MS if an individual has suffered separate episodes of neurologic symptoms characteristic of MS. Since some people seek medical attention after only one attack, other testing may hasten and ease the diagnosis. The most commonly used diagnostic tools are neuroimaging, analysis of cerebrospinal fluid, and evoked potentials. The MRI brain and spine shows the areas of demyelination (lesions or plaques). Gadolinium can be administered intravenously as a contrast to highlight active plaques and, by elimination, demonstrate the existence of historical lesions not associated with symptoms at the moment of the evaluation. Testing of CSF obtained from a lumbar puncture can provide evidence of chronic inflammation of the CNS. The cerebrospinal fluid is tested for oligoclonal bands of IgG on electrophoresis, which are inflammation markers found in 75–85% of people with MS. The nervous system of a person with MS responds less actively to stimulation of the optic nerve and sensory nerves due to demyelination of such pathways. These brain responses can be examined using visual and sensory evoked potentials.
 
Progression of Multiple Sclerosis Subtypes
Several subtypes, or patterns of progression, have been described. Subtypes use the past course of the disease in an attempt to predict the future course. They are important not only for prognosis but also for therapeutic decisions. In 1996, the United States National Multiple Sclerosis Society standardized four clinical courses:
  1. Relapsing remitting
  2. Secondary progressive
  3. Primary progressive
  4. Progressive relapsing.
The relapsing remitting subtype is characterized by unpredictable relapses followed by periods of months to years of relative quiet (remission) with no new signs of disease activity. Deficits suffered during attacks may either resolve or leave sequelae, the latter being more common as a function of time. This describes the initial course of 80% of individuals with MS. When deficits always resolve between attacks, this is sometimes referred to as benign MS, although people will still accrue some degree of disability in the long-term. The relapsing remitting subtype usually begins with a clinically isolated syndrome (CIS). In CIS, a person has an attack suggestive of demyelination, but does not fulfill the criteria for MS. However, only 30–70% of persons experiencing CIS later develop MS.
Secondary progressive MS (sometimes called “galloping MS”) describes around 65% of those with an initial relapsing remitting MS, who then begin to have progressive neurologic decline between acute attacks without any definite periods of remission. Occasional relapses and minor remissions 77may appear. The median time between disease onset and conversion from relapsing remitting to secondary progressive MS is 19 years. The primary progressive subtype describes the approximately 10–15% of individuals who never have remission after their initial MS symptoms. It is characterized by progression of disability from onset, with no, or only occasional and minor, remissions and improvements. The age of onset for the primary progressive subtype is later than for the relapsing remitting, but similar to mean age of progression between the relapsing remitting and the secondary progressive. In both cases, it is around 40 years of age.
Progressive relapsing MS describes those individuals who, from onset, have a steady neurologic decline but also suffer clear superimposed attacks. This is the least common of all subtypes.
Atypical variants of MS with nonstandard behavior have been described: these include Devic's disease, Balo concentric sclerosis, Schilder's diffuse sclerosis, and Marburg MS. There is debate on whether they are MS variants or different diseases. Multiple sclerosis also behaves differently in children, taking more time to reach the progressive stage. Nevertheless, they still reach it at a lower mean age than adults.
 
Management
Although there is no known cure for MS, several therapies have proven helpful. The primary aims of therapy are returning function after an attack, preventing new attacks, and preventing disability. As with any medical treatment, medications used in the management of MS have several adverse effects. Alternative treatments are pursued by some people, despite the shortage of supporting, comparable, replicated scientific study.
 
Acute Attacks
During symptomatic attacks, administration of high doses of intravenous corticosteroids, such as methylprednisolone, is the routine therapy for acute relapses. Although generally effective in the short-term for relieving symptoms, corticosteroid treatments do not appear to have a significant impact on long-term recovery. Oral and intravenous administration seems to have similar efficacy. Consequences of severe attacks, which do not respond to corticosteroids, might be treated by p lasmapheresis.
 
Disease-modifying Treatments
Disease-modifying treatments are expensive, and most of these require frequent (up to daily) injections. Others require IV infusions at 1–3 months intervals.
Fingolimod (trade name Gilenya) was approved for MS by the FDA in 2010, and in Europe in 2011. As of 2011 (update), after this approval, there are six disease-modifying treatments for MS approved by regulatory agencies of various countries, being the other five: Interferon β 1a (trade names Avonex, CinnoVex, ReciGen, and Rebif) and interferon β 1b (US trade name Bseron, in Europe and Japan Bferon). A third medication is glatiramer 78acetate (Copaxone), a noninterferon, nonsteroidal immunomodulator. The fourth medication, mitoxantrone, is an immunosuppressant also used in cancer chemotherapy. The fifth is a humanized monoclonal antibody immunomodulator, natalizumab (marketed as Tysabri). The interferons and glatiramer acetate are delivered by frequent injections, varying from once per day for glatiramer acetate to once per week (but intramuscular) for Avonex. Natalizumab and mitoxantrone are given by IV infusion at monthly intervals.
All six kinds of medications are modestly effective at decreasing the number of attacks in relapsing remitting MS (RRMS), while the capacity of interferons and glatiramer acetate is more controversial. Studies of their long-term effects are still lacking. Comparisons between immunomodulators (all but mitoxantrone) show that the most effective is natalizumab, both in terms of relapse rate reduction and halting disability progression. Mitoxantrone may be the most effective of them all; however, it is generally not considered as a long-term therapy, as its use is limited by severe secondary effects. The earliest clinical presentation of RRMS is the CIS. Treatment with interferons during an initial attack can decrease the chance that a person will develop clinical MS.
Treatment of progressive MS is more difficult than relapsing remitting MS. Mitoxantrone has shown positive effects in those with secondary progressive and progressive relapsing courses. It is moderately effective in reducing the progression of the disease and the frequency of relapses in short term follow up. No treatment has been proven to modify the course of primary progressive MS.
As with many medical treatments, these treatments have several adverse effects. One of the most common is irritation at the injection site for glatiramer acetate and the interferon treatments. Over time, a visible dent at the injection site due to the local destruction of fat tissue, known as lipoatrophy, may develop. Interferons produce symptoms similar to influenza. Some people taking glatiramer experience a post-injection reaction manifested by flushing, chest tightness, heart palpitations, breathlessness, and anxiety, which usually lasts less than thirty minutes. More dangerous but much less common are liver damage from interferons, severe cardiotoxicity, infertility, and acute myeloid leukemia of mitoxantrone, and the putative link between natalizumab, and some cases of progressive multifocal leukoencephalopathy.
 
Management of the Effects of Multiple Sclerosis
Disease-modifying treatments reduce the progression rate of the disease but do not stop it. As MS progresses, the symptomatology tends to increase. The disease is associated with a variety of symptoms and functional deficits, which result in a range of progressive impairments and disability. Management of these deficits is therefore very important. Both drug therapy and neurorehabilitation have shown to ease the burden of some symptoms, though neither influences disease progression. Some symptoms have a good response to medication, such as unstable bladder and spasticity, while management of many others is much more complicated. As for any person with neurologic deficits, a multidisciplinary approach is the key to improving 79quality of life; however, there are particular difficulties in specifying a “core team” because people with MS may need help from almost any health profession or service at some point. Multidisciplinary rehabilitation programs increase activity and participation of people with MS but do not influence impairment level.
Historically, individuals with MS were advised against participation in physical activity due to worsening symptoms. However, under the direction of a physiotherapist, participation in physical activity can be safe and has been proven beneficial for persons with MS. Research has supported the rehabilitative role of physical activity in improving muscle power, mobility, mood, bowel health, general conditioning and quality of life. Care should be taken not to overheat a person with MS during the course of exercise. Physiotherapists have the expertise needed to adequately prescribe exercise programs, which suitable for the individual. The frequency of exercise, intensity of exercise, type of exercise and time/duration of exercise (FITT equation) is typically used to prescribe exercises. Depending on the person, activities may include resistance training, walking, swimming, yoga, tai chi, and others. Determining an appropriate and safe exercise program is challenging and must be carefully individualized to each person being sure to account for all contraindications and precautions. There is some evidence that cooling measures are effective in allowing a greater degree of exercise.
 
Alternative Treatments
Many people with MS use complementary and alternative medicine. Depending on the treatments, the evidence is weak or absent. Examples are a dietary regimen, herbal medicine (including the use of medical cannabis), hyperbaric oxygenation and self infection with hookworm (known generally as helminthic therapy).
 
Prognosis
The prognosis (the expected future course of the disease) for a person with MS depends on the subtype of the disease; the individual's sex, age, and initial symptoms; and the degree of disability the person experiences. The disease evolves and advances over decades, 30 being the mean years to death since onset.
Female sex, relapsing remitting subtype, optic neuritis or sensory symptoms at onset, few attacks in the initial years, and especially, early age at onset are associated with a better course.
The life expectancy of people with MS is 5–10 years lower than that of unaffected people. Almost 40% of people with MS reach the seventh decade of life. Nevertheless, two-thirds of the deaths in people with MS are directly related to the consequences of the disease. Suicide also has a higher prevalence than in the healthy population, while infections and complications are especially hazardous for the more disabled ones.
Although most people lose the ability to walk prior to death, 90% are still capable of independent walking at 10 years from onset, and 75% at 15 years. 80
 
Epidemiology
Two main measures are used in epidemiological studies: incidence and prevalence. Incidence is the number of new cases per unit of person–time at risk (usually number of new cases per thousand person–years), while prevalence is the total number of cases of the disease in the population at a given time. Prevalence is known to depend not only on incidence, but also on survival rate and migrations of affected people. MS has a prevalence that ranges between 2 and 150 per 100,000 depending on the country or specific population. Studies on population and geographical patterns of epidemiological measures have been very common in MS and have led to the proposal of different etiological (causal) theories.
Multiple sclerosis usually appears in adults in their thirties, but it can also appear in children. The primary progressive subtype is more common in people in their fifties. As with many autoimmune disorders, the disease is more common in women, and the trend may be increasing. In children, the sex ratio difference is higher, while in people over fifty; MS affects males and females almost equally.
There is a north to south gradient in the northern hemisphere and a south to north gradient in the southern hemisphere, with MS being much less common in people living near the equator. Climate, sunlight and intake of vitamin D have been investigated as possible causes of the disease that could explain this latitude gradient. However, there are important exceptions to the north-south pattern and changes in prevalence rates over time; in general, this trend might be disappearing. This indicates that other factors, such as environment or genetics have to be taken into account to explain the origin of MS.
Environmental factors during childhood may play an important role in the development of MS later in life. Several studies of migrants show that if migration occurs before the age of 15, the migrant acquires the new region's susceptibility to MS. If migration takes place after age 15, the migrant retains the susceptibility of his home country. However, the age-geographical risk for developing MS may span a larger timescale. A relationship between season of birth and MS has also been found, which lends support to an association with sunlight and vitamin D; for example, fewer people with MS are born in November as compared to May.
 
Therapies
Research directions on MS treatments include investigations of MS pathogenesis and heterogeneity; research of more effective, convenient, or tolerable new treatments for RRMS; creation of therapies for the progressive subtypes; neuroprotection strategies; and the search for effective symptomatic treatments. A number of treatments, which may curtail attacks or improve function are under investigation. Emerging agents for RRMS that have shown promise in phase 2 trials include alemtuzumab (trade name Campath), daclizumab (trade name Zenapax), rituximab, dirucotide, 81BHT 3009, cladribine, dimethyl fumarate, estriol, laquinimod, PEGylated interferon β 1a, minocycline, statins, temsirolimus and teriflunomide.
In 2010, an FDA committee recommended approving fingolimod for the treatment of MS attacks, and on September 22, 2010, fingolimod (trade name Gilenya) became the first oral drug approved by the Food and Drug Administration to reduce relapses and delay disability progression in people with relapsing forms of MS. Clinical trials of fingolimod have demonstrated side effects, including cardiovascular conditions, macular edema, infections, liver toxicity, and malignancies.
Much interest has been focused on the prospect of utilizing vitamin D analogs in the prevention and management of CIS and MS, especially given its possible role in the pathogenesis of the disease. While there is anecdotal evidence of benefit for low dose naltrexone, only results from a pilot study in primary progressive MS have been published.
 
Disease Biomarkers
The variable clinical presentation of MS and the lack of diagnostic laboratory tests lead to delays in diagnosis and the impossibility of predicting diagnosis. New diagnostic methods are being investigated. These include work with antimyelin antibodies, analysis of microarray gene expression and studies with serum and cerebrospinal fluid, but none of them has yielded reliable positive results.
Currently there are no clinically established laboratory investigations available, which can predict prognosis. However, several promising approaches have been proposed. Investigations on the prediction of evolution have centered around monitoring disease activity. Disease activation biomarkers include interleukin 6, nitric oxide and nitric oxide synthase, osteopontin, and fetuin A. On the other hand, since disease progression is the result of neurodegeneration, the roles of proteins indicative of neuronal, axonal, and glial loss, such as neurofilaments, tau and N acetylaspartate are under investigation.
A final investigative field is work with biomarkers that distinguish between medication responders and nonresponders.
 
Chronic Cerebrospinal Venous Insufficiency
In 2008, Italian vascular surgeon Paolo Zamboni reported research suggesting that MS involve a vascular disease process he referred to as chronic cerebrospinal venous insufficiency (CCSVI), in which veins from the brain were constricted. He found CCSVI in the majority of people with MS, performed a surgical procedure to correct it and claimed that 73% of people improved. Concern has been raised with Zamboni's research as it was neither blinded nor controlled, and further studies have had variable results. This has raised serious objections to the hypothesis of CCSVI originating MS. The neurology community currently recommends not using the proposed treatment unless its effectiveness is confirmed by controlled studies, the 82need for which has been recognized by the scientific bodies engaged in MS research.
 
Syndromes of Non-disseminated Demyelination
A number of disorders are clearly related to MS, while the remaining are distinct. Some, such as optic neuritis, appear pathologically identical, but are disseminated in neither time nor space. Whether they herald MS is plainly of huge importance to patients, who are now sufficiently informed to know of and fear this possibility. There is here a lacune in the neurological lexicon. It would be helpful to call upon a collective term for acute monophasic and monofocal syndromes that are seen in MS idiopathic optic neuritis, sensory myelitis, and many others (after including, one suspects, episodes dismissed as labyrinthitis, trigeminal neuralgia, and Bell's palsy) but which do not develop into MS.
Other syndromes, such as acute transverse myelitis, show distinctive pathological features and represent (often clinically recognizable) separate disorders.
 
OPTIC NEURITIS
The clinical syndrome of optic neuritis is not always a manifestation of idiopathic inflammatory demyelination; and, even when it is, does not invariably presage MS itself. Identifiable primary causes include Leber's hereditary optic neuropathy (LHON), typically causing bilateral simultaneous or rapidly sequential optic neuritis (unusual in MS, and a feature which should prompt a more intense search for primary causes), which is very severe (and usually permanent) in young men. LHON may be spotted by careful fundoscopy (preferably using a slit lamp), showing tortuous vessels with capillary dilatation and telangiectasia, without increase in vascular permeability apparent on fluorescein angiography, contrasting with optic neuritis. These changes are not invariable, and mitochondrial DNA analysis is the proper route of diagnostic confirmation.
The differential diagnosis also includes:
  • Toxins (most notoriously tobacco amblyopia and methanol)
  • Vitamin B12 deficiency
  • Other inflammatory disorders (particularly sarcoidosis, vasculitis, and lupus
  • Infections (uncommon)
  • Ischemia (less uncommon), usually revealing itself by a typically vascular more abrupt onset, an absence of pain, and a horizontal altitudinal field defect
  • Optic nerve, chiasmal, or other local tumors these may be intrinsic (classically gliomata) or optic nerve sheath meningiomata, the latter suggested by the presence of optociliary shunt vessels or extrinsic (pituitary and craniopharyngiomata).
There are no useful clinical clues indicating whether or not optic neuritis in any one individual heralds MS. An MRI scanning serves multiple functions: directly to disclose optic neuritis, to help exclude many of the 83above alternatives, to help prognosticate long and/or intracanalicular lesions are associated with poorer visual recovery, and to help predict the future risk of MS. Multifocal white matter lesions are seen at presentation in 50–70% of cases. Their presence indicates a risk of 82% in 5 years, while a normal brain MRI carries a predictive risk of between 6% and 24% at 5 years.
 
Transverse Myelitis
“Idiopathic” transverse myelitis usually exhibits a rather different clinical phenotype to the spinal cord relapse of MS. In approximately 80% of cases, the thoracic cord is affected, and as the name suggests, the usual clinical picture suggests involvement of the whole transverse extent of the spinal cord. The usual picture is, therefore, one of rapidly progressing paralysis, sensory loss and incontinence, often with back pain; fever and meninges may be present. Objectively, the flaccid paraparetic or paraplegic picture of spinal shock with useless bladder and/or bowel sphincters is found, often with a sensory level accompanied by a band of hyperesthesia, allodynia or hyperpathia. In MS, partial cord lesions are much more typical pure sensory disturbance in both legs, and deafferentation of one arm. Spinal shock is quite uncommon.
A history of preceding infection (usually respiratory) in approximately one-third of cases helps emphasize a closer affinity with acute disseminated encephalomyelitis (ADEM) than with MS, a relation further substantiated by a more destructive histopathological picture. MR scanning reveals a more destructive inflammatory process, far more extensive longitudinally than the clinical picture (or the name) implies. Primary causes—infections, including zoster, retroviruses, both HIV and human T-lymphotropic virus (HTLV) 1, and systemic inflammatory disorders, most notoriously SLE, must be considered. SLE can cause a severe acute myelopathy. Spinal cord ischemia, unless signposted by an obvious precipitant (an expanding and/or dissecting aortic aneurysm, for example) can be more difficult to rule out with any certainty.
As with ADEM, spinal fluid analysis in transverse myelitis may reveal an increased mononuclear cell count and protein concentration, but oligoclonal band testing is positive only in a minority of cases, providing some help in the distinction from MS.
High dose intravenous corticosteroids are commonly administered, but the prognosis is at best middling.
 
Devic's Disease or Neuromyelitis Optica
It is an acute or subacute optic neuritis associated with myelitis, which occurs in at least three separate contexts. It may be seen in MS, if the burden of disease happens to fall asymmetrically upon the spinal cord and optic nerves. In this situation, conventionally disseminated lesions are apparent elsewhere in the CNS, disclosed by MRI. Secondly, other inflammatory disorders may not only cause a similar clinical picture, notoriously SLE, but also vasculitic syndromes, sarcoidosis and Behçet's disease. Usually, but not invariably, systemic or serological manifestations are apparent. There is, in addition, a group of 84patients with no clinical or paraclinical evidence of disease elsewhere in the CNS, and in whom other systemic or vasculitic disorders have been excluded. The diagnostic label of Devic's disease should be reserved for these, and they show a unique clinical and pathological phenotype clearly separable from MS. Oligoclonal bands are usually absent and cranial MRI is normal. The pathological process is closer to that of transverse myelitis or ADEM.
 
Foix-Alajouanine Syndrome
In 1926, Foix and Alajouanine described subacute necrotic myelitis, an illness predominantly affecting adult males, characterized by a spastic paralysis, sensory loss, and incontinence progressing over 2–3 months; a flaccid, areflexic, amyotrophic phase ensued. Signs of systemic illness, with fever, meningism, and often severe local pain, were common. Spinal fluid changes (cytoalbuminic dissociation with greatly increased protein concentrations) and, in later cases, imaging, indicated spinal block from a very swollen cord. A number of reports followed describing patients with similarities, but often more reactive CSF, and the clinical picture has been left rather confused (whether the disorder is primarily inflammatory or not varies with different authorities).
Most neuropathologists, perhaps faithful to its originators, retain the eponym for a disorder characterized by a severe necrotic process, of putative veno-occlusive cause, affecting gray and white matter, often with thrombin deposition thickening blood vessel walls.
 
Diffuse or Disseminated Syndromes
 
Acute Disseminated Encephalomyelitis
It occurs predominantly, though by no means exclusively, in childhood perhaps this is because the most common viral precipitants of postinfectious encephalomyelitis are the childhood exanthemata, ADEM also occurs as postvaccination encephalomyelitis, but in a proportion of cases, no antecedent immunological challenge is identifiable.
Typically, between days and 2–3 weeks following a self-limiting illness, there emerges a prodrome of fever, myalgia and malaise, lasting a few days. Subsequently, acutely evolving neurological features occur whose nature indicates severe simultaneous or rapidly sequential multifocal CNS disease. Focal brainstem and/or hemisphere signs, transverse myelitis, and cranial neuropathies, including bilateral optic neuritis (unilateral disease is uncommon in this context), occur. Cerebellar ataxia is particularly associated with varicella (and a good prognosis).
Less focally, there may occur an encephalopathy (which may progress to coma), meningism, and seizures. These, the bilateral nature of optic neuritis and the multifocal nature of the disorder (acute episodes in MS are usually symptomatically single sited), all suggest ADEM rather than MS. Uncommonly, ADEM can relapse persistently, rendering the distinction from MS very difficult indeed, though so called multiphasic disseminated encephalomyelitis (MDEM) probably represents a distinct disorder. The 85spinal fluid, often cellular, usually contains no oligoclonal bands. Multifocal MRI lesions are observed, but are often more extensive and symmetrical in the white matter and, occasionally, in the basal ganglia than in MS. Gadolinium enhancement can also help to distinguish the disorders—a mixture of enhancing and nonenhancing lesions implies the temporal dissemination of MS. Although spontaneous recovery is the rule, a fatal outcome is seen in approximately 10–20%.
Acute hemorrhagic leukoencephalomyelitis or Weston Hurst disease is a rare, more severe, and commonly fatal hyperacute form of ADEM. The course is more rapid, with pronounced systemic features: seizures are frequent and coma usual. CSF analysis often reveals a raised intracranial pressure, and a pleomorphic cellular reaction with lymphocytes, neutrophils and significant numbers of red cells, reflecting the microhemorrhagic process.
 
Apparent Variants of Multiple Sclerosis
During the early part of this century, a number of disorders were described which are now considered clinicopathological variants of MS.
Marburg disease essentially represents MS with a strikingly aggressive or malignant course, often rapidly fatal, sometimes occurring after one or two isolated demyelinating events. Retention of the term is useful on clinical grounds.
Schilder's diffuse sclerosis is more complex. Dr Schilder certainly found it difficult to recognize, his second report describing two patients who had other diseases. However, there is a syndrome distinct from, but related to MS, usually of childhood onset, whose progressive course is punctuated by occasional periods of accelerated disease activity. Widespread, confluent or diffuse demyelinations involving the cerebrum, cerebellum, and brainstem, with pronounced axon loss and often cavitation, are characteristic features. The neurological manifestations include those seen in MS, although dementia and other mainly cortical features are more prominent, including hemiplegia, cortical blindness, and deafness. Inherited leukodystrophies (see below) must be excluded.
Balò's concentric sclerosis is characterized pathologically by alternating concentric rings of demyelination and apparently normal myelin occasionally strikingly seen on MRI. Episodes of repeated inflammatory demyelination at the same site, with intervening periods of myelin repair, probably explain this architecture. No particular clinical phenotype is commonly associated.
 
Inherited Disorders Which May Be Confused with Multiple Sclerosis
Adrenoleukodystrophy and metachromatic leukodystrophy may both cause a picture resembling progressive MS. Slowly progressive disease is the rule, but remitting illness is recognized. A family history, abdominal symptoms, skin pigmentation, or other Addisonian features and absent oligoclonal bands 86should stimulate a search for very long chain fatty acids and/or leukocyte enzyme abnormalities; additionally, MRI in leukodystrophies usually has partial specificity. Female carriers of the adrenoleukodystrophy gene can manifest. Electrophysiological evidence of a peripheral neuropathy also helps point to these disorders when confusion arises.
Mitochondrial disease can also cause a relapsing–remitting multifocal neurological picture. Useful distinguishing clinical features are found, and oligoclonal bands are mostly absent. Leber's disease is usually readily distinguished from MS. However, an interesting variant has emerged in recent years. Females present with disease consistent with MS, but with a particular burden on the optic nerves; CSF oligoclonal bands and cranial MRI changes suggest MS, but genetic tests reveal the presence of Leber's mitochondrial mutations.
 
INFECTIOUS DISEASES
Human T-lymphotropic virus 1 related myelopathy is mentioned below. Lyme disease can cause a phenotype very similar to MS. The more common acute picture of meningism (or meningoencephalitis), facial palsy and/or painful radiculopathy presents few problems. However, in so called tertiary Lyme disease, progressive syndromes, including spastic paraparesis, cerebellar ataxia, and recurrent cranial neuropathies can cause diagnostic confusion. MRI may show multifocal white matter lesions, and the CSF can contain oligoclonal bands, though the cell count is usually persistently high.
A history of the characteristic skin rash several years earlier will not be mentioned unless directly queried (and occasionally not even then). Serological tests on both blood and CSF are therefore important. Polymerase chain reaction (PCR) also now plays a role. Neurosyphilis, while increasing in frequency, still earns mention mostly through historic respect; it is rarely confused with MS, particularly in the MRI era. Headache and fits are common and pupillary abnormalities characteristic. Syphilitic optic neuritis is usually painless.
 
Unrelated Inflammatory Disorders
Neurological disturbance reflecting involvement of the nervous system in many systemic inflammatory diseases can mimic MS.
 
Cerebral Vasculitis
Vasculitis is a histopathological description, not a diagnosis or disease. In both isolated CNS vasculitis and CNS involvement in systemic vasculitis three broad clinical phenotypes are proposed. That specifically resembling MS (MS plus) exhibits a relapsing—remitting course, with features, such as optic neuropathy and brainstem episodes. However, additional features less common in MS also occur seizures, severe persisting headaches (said to occur in 80% of patients or more), encephalopathic episodes or hemispheric stroke like events. Systemic features may also be present (often revealed only on direct inquiry) even in so called isolated CNS vasculitis fever and night 87sweats, skin or eye changes, oligoarthropathy also contrasting with MS. The other two clinical patterns are:
  1. Acute or Subacute encephalopathy.
  2. Mass lesion (these are of no known pathological or therapeutic moment, but may help improve recognition).
Once suspected, diagnosis or exclusion of isolated/primary cerebral vasculitis can be difficult (the context of known systemic disease plainly does not pose this problem). Serological markers, including ANCA (antineutrophil cytoplasmic antibody) should be sought but are commonly negative. Spinal fluid examination is like ESR testing often abnormal (in 65–80% of cases) but lacks specificity, with changes in cell count, protein, and/or immunoglobulin band analysis. MRI can closely resemble MS, but may be normal. Contrast angiography may show segmental (often multifocal) narrowing and areas of localized dilatation or beading, often with areas of occlusion, also rarely with aneurysms. While these changes are also nonspecific, they are not seen in MS. However, the false-negative rate for angiography is 30–80%. Therefore, histopathological confirmation, either by taking a biopsy of a lesion if possible, or by “blind” biopsy incorporating meninges and nondominant temporal white and gray matter, can be important, though not a trivial procedure (carrying a 0.5–2% risk of serious morbidity). The distinction is important, as cyclophosphamide with steroids is of value in the management of confirmed vasculitis.
 
Systemic Lupus Erythematosus
Neurological involvement in SLE is seen in 50% of cases, but neurological presentation is found in perhaps only 3% as with vasculitis, neurological disease in the setting of known lupus presents less of a problem. It is uncommon therefore for lupus and MS to be confused diagnostically. This said the historic concept of so called lupoid sclerosis, MS like neurological features in the context of established lupus, needs to be mentioned in order to be dismissed. Pathological studies indicate that primary demyelination is not seen in CNS lupus, helping to emphasize the separateness of these disorders. The simple coexistence of lupus and MS, neither being excessively rare, may have contributed to this confusion, as has the over interpretation of antinuclear antibody (ANA) serology.
Direct inquiry and focused systemic examination to disclose fever, malaise, skin changes (classically, the malar butterfly rash and/or photosensitivity) (Figs 11A and B), and arthritis will help. Revised SLE diagnostic criteria, with an estimated specificity and sensitivity of 96%, have been widely accepted, particularly for research and therapeutic trial purposes. Importantly, most authorities suggest that only ANA titers over 1:160 are diagnostically relevant.
Again in common with vasculitis, a wide variety of CNS complications can occur in SLE, some very similar to those in MS, others pointing away from this diagnosis. CNS lupus very rarely has an underlying vasculitic pathology, and the term lupus vasculitis should also be discarded.
Ataxia, brainstem abnormalities, and cranial neuropathies may resemble MS, but more particularly associated with lupus are optic neuropathy and 88transverse myelopathy.
Figs 11A and B: A. Systemic lupus erythematosus. B. Malar butterfly rash
The former is often painless, subacute and progressive, and commonly very severe; the latter usually resembles idiopathic transverse myelitis more than spinal relapses of MS. Headache is common in lupus (including that of dural sinus thrombosis). Other features for which SLE is more noted for than MS include seizures, psychiatric and cognitive disturbances, episodes of encephalopathy, and movement disorders (especially chorea). Peripheral neuropathy can also occur. Stroke, particularly a feature of the antiphospholipid syndrome, rarely causes diagnostic confusion with MS.
 
Sjogren's Syndrome
Characteristically comprises (i) keratoconjunctivitis sicca, and (ii) xerostomia (these occurring in approximately 50% of cases), in the context of (iii) another connective tissue disorder, usually rheumatoid arthritis. Speckled anti Ro (SS-A) or anti La (SS-B) antibodies are present in up to 75–80% of patients. The principal neurological manifestations are peripheral. Trigeminal sensory neuropathy is classically described.
More recently, attention has been drawn to CNS complications, and particularly to an MS-like picture (optic neuropathy is particularly associated). Symptoms of dry mouth and eyes should routinely be sought, although as with SLE, peripheral features such as neuropathy or myositis (Figs 12A and B), or CNS disturbances, including seizures, stroke like neurological deficits, an encephalopathy with or without an aseptic meningitis, and/or psychiatric abnormalities in addition to the systemic features mean that MS is rarely confused.
 
Neurosarcoidosis
Sarcoidosis affects the nervous system in approximately 5% of patients. Optic nerve disease is particularly associated; a chronic progressive course and persistent steroid sensitivity commonly (but not invariably) point away from MS. Other cranial neuropathies (especially involving the facial nerve), and brainstem and spinal cord disease, may variably resemble MS. Cognitive and neuropsychiatric abnormalities, and peripheral involvement (nerve and muscle) help point away from MS.89
Figs 12A and B: A. Sjogren's syndrome; B. Marked bilateral parotid gland, dry mouth resulting in increased tooth decay
The chest X-ray is abnormal in between a third and a half of patients; subclinical thoracic disease is said to be present in most cases of extrathoracic sarcoidosis. Searching for anterior and/or posterior segment inflammation using slit lamp examination and fluorescein angiography can be valuable. Serum and CSF angiotensin converting enzyme concentrations may be raised; the CSF may reveal increases in protein or cell count in 80% of cases. Oligoclonal bands may be present, though as with other non-MS pathologies, their presence varies when serially assessed. Whole body gallium scanning can disclose asymptomatic foci of systemic disease. Cranial MRI may show multiple white matter lesions and/or, in about one-third of patients, meningeal enhancement. The diagnosis is confirmed where possible by biopsy, either of cerebral or meningeal tissue, or of lung or conjunctiva where appropriate.
 
Behçet's Disease
It is a chronic relapsing multisystem inflammatory disorder. Formal diagnostic criteria propose that recurrent oral ulceration (at least three times in one year) is an absolute criterion; any two of (i) recurrent genital ulceration, (ii) uveitis or retinal vasculitis, (iii) skin lesions (Figs 13A and B), including erythema nodosum or acneiform nodules, pseudofolliculitis or papulopustular lesions, or (iv) a positive pathergy test (read at 24–48 hours) are also required to confirm the diagnosis. These again help emphasize the importance of a careful directed history and examination in revealing crucial features, which patients might not think of sufficient interest to the neurologist to mention.
If benign headache is excluded, approximately 5% of patients develop neurological complications. Features suggesting Behçet's disease include cerebral venous sinus thrombosis, sterile meningoencephalitis, encephalopathy, and psychiatric and progressive cognitive manifestations. MRI abnormalities are nonspecific, though posterior fossa and brainstem involvement is said to be typical. Oligoclonal bands are uncommon.90
Figs 13A and B: Skin problems are a common symptom of Behcet's disease
 
Whipple's Disease
Caused by Tropheryma whippelii, is characterized by arthropathy, respiratory symptoms, anemia, fever, erythema nodosum, and severe wasting, in addition to steatorrhea and abdominal distension. Ten percent of patients have neurological involvement, and 5% present in this way.
Imaging may be normal, or reveal nonspecific abnormalities, and the same may be said of the CSF. One-third of CSF samples may reveal pathognomic period acid Schiff (PAS) positive bacilli. Repeat spinal fluid examination increases this yield. Approximately 30% of cases have a noninformative small bowel biopsy though electron microscopy will increase sensitivity. Lymph node biopsy can also be useful. PCR analysis of blood, lymph node, spinal fluid, small bowel tissue or brain is increasingly used. Neurological disease may be reversible if treated promptly (with tetracycline, penicillin or more commonly, co-trimoxazole), or rapidly fatal if not.
 
Vascular Disease
Arteriovenous malformations enjoyed certain notoriety as a confounding cause of a relapsing remitting single sited syndrome, but because of the availability of MRI, they no longer command such diagnostic respect. Subacute bacterial endocarditis (SBE) and atrial myxoma can rarely cause a more challenging picture as a consequence of multifocal embolic infarction, but the context is usually distractingly obvious. Cerebral autosomal dominant arteriopathy with subcortical and leukoencephalopathy (CADASIL) can mislead, but the family history, cognitive features, usually distinctive MRI changes, and absence of oligoclonal bands should alert the tolerably wary.
At the end diseases, such as subacute sclerosing panencephalitis, herpes simplex encephalitis, meningeal carcinomatosis, meningoencephalitis, neurosyphilis, Guillain-Barré syndrome among others cannot be definitively differentiated from MS through electrophoresis.91
The absence of diagnostic tests means that uncertainty can be extremely difficult to resolve. MRI, spinal fluid examination, and evoked potential recordings are sensitive tests for MS but do not have comparable specificity. The range of disorders, which can mimic MS is vast; as the prototypical inflammatory demyelinating disorder, it may be confused with both unrelated demyelinating diseases (metabolic or inherited) and unrelated inflammatory disease. Additionally, diseases, which are directly related to MS must also be considered. To offer a systematic account of all would be unrealistic, and not a little unreadable. Therefore, only a few comments relating to salient clinical features or discriminating investigations will be mentioned.
Clinical significance of proteinurias electrophoresis: Proteinuria is defined as urinary protein excretion of more than 150 mg per day. Microalbuminuria can be a sign of early renal disease, especially in diabetic patients. In microalbuminuria, 30–150 mg of protein per day is excreted per day.
Detection and identification of proteinuria is helpful information in the diagnosis of renal failure. Proteinuria may result from many pathological conditions. Identification of the main proteins excreted into urine helps in pinpointing the type of the kidney damage (tubular, glomerular, or mixed) and in diagnosis of the underlying pathology (Bence Jones proteins).
Immunofixation electrophoresis allows the proteins to be anchored in situ after electrophoresis, by forming insoluble complexes with corresponding antisera. The procedure allows a single step characterization of the various proteinuria profiles and detection of Bence Jones proteins as a qualitative aid in the identification of monoclonal gammopathies.
Proteinuria can be transient (intermittent), orthostatic (relate to sitting/standing) or persistent (always present). In transient proteinuria, protein in urine disappears when the underlying cause is resolved. In orthostatic proteinuria, protein excretion is normal when the patient is lying down but is increased when a person is sitting or standing. It occurs in approximately 2–5% of young people but is unusual in people over the age of 30 years.
Persistent proteinuria can be further defined as glomerular, tubular, or overflow. The most common type is glomerular proteinuria with albumin as the primary urinary protein. This type of proteinuria is caused by increased filtration of albumin and other macromolecules across the glomerular basement membrane.
Tubular proteinuria results when malfunctioning tubule cells no longer reabsorb proteins in the filtrate. Low-molecular weight proteins, such as β2 microglobulin and immunoglobulin light chains dominate over albumin.
In overflow proteinuria, low-molecular weight proteins overwhelm the ability of the tubules to reabsorb the filtered proteins. Bence Jones proteinuria is the classic example of overflow (and also tubular) proteinuria.
Quantitative measurement of Bence Jones proteins, and determination that they are monoclonal, aid in the diagnosis of various disorders, including multiple myeloma—the malignant proliferation of plasma cells.92
Urine protein electrophoresis (UPEP) is utilized to detect monoclonal and other proteins in urine. The test provides information about the location and degree of damage within the nephron.
The type of testing, the sensitivity of the protein stain, and the total protein concentration in the sample dictate whether the urine needs to be concentrated or run neat. Urine immunofixation electrophoresis (UIFE) helps determine the type of protein detected by UPEP.
The following conditions warrant urine protein electrophoresis:
  • Monoclonal protein in serum is more than 1.5 g/dL
  • Monoclonal free light chains are detected in serum
  • Hypogammaglobulinemia is present in serum
  • Serum electrophoresis shows nephrotic pattern.
Recently, through a prospective study, Giovanni et al. proved the importance of urinary studies in patients with suspected amyloidosis.
 
AMYLOIDOSIS
Refers to a variety of conditions wherein amyloid proteins are abnormally deposited in organs or tissues and cause harm. A protein is described as being amyloid if, due to an alteration in its secondary structure, it takes on a particular aggregated insoluble form, similar to the β-pleated sheet. Symptoms vary widely depending upon where in the body amyloid deposits accumulate. Amyloidosis may be inherited or acquired.
The modern classification of amyloid disease tends to use an abbreviation of the protein that makes the majority of deposits, prefixed with the letter A. For example, amyloidosis caused by transthyretin is termed “ATTR”. Deposition patterns vary between patients but are almost always composed of just one amyloidogenic protein. Deposition can be systemic (affecting many different organ systems) or organ specific. Many amyloidosis are inherited, due to mutations in the precursor protein.
Other forms are due to different diseases causing overabundant or abnormal protein production, such as with overproduction of immunoglobulin light chains in multiple myeloma (termed AL amyloidosis), or with continuous overproduction of acute phase proteins in chronic inflammation.
 
Pathogenesis
When a native cell creates a protein, it could either make the actual protein or protein fragments. These fragments could come and join together to form the actual protein. Such a protein can sometimes regress into the protein fragments. This process of “flip flopping” happens frequently in certain proteins, especially the ones that cause this disease.
The fragments or actual proteins are at risk of misfolding as they are synthesized to make a bad protein. This causes proteolysis, which is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion. Proteases come and digest the misfolded 93fragments and proteins. The problem occurs when the proteins do not dissolve in proteolysis. This happens because the misfolded proteins sometimes become robust enough that they are not dissolved by normal proteolysis. When the fragments do not dissolve, they get spit out of proteolysis, and they aggregate to form oligomers. The reason they aggregate is that the parts of the protein that do not dissolve in proteolysis are the β-pleated sheets, which are extremely hydrophobic. They are usually sequestered in the middle of the protein, while parts of the protein that are more soluble are found near the outside. When they are exposed to water, these hydrophobic pieces tend to aggregate with other hydrophobic pieces. This ball of fragments gets stabilized by GAG's (glycosaminoglycans) and SAP (serum amyloid P), a component found in amyloid aggregations that is thought to stabilize them and prevent proteolytic cleavage. The stabilized balls of protein fragments are called oligomers. The oligomers can aggregate together and further stabilize to make amyloid fibrils.
Both the oligomers and amyloid fibrils can cause cell toxicity and organ dysfunction.
The names of the amyloid usually start with the letter “A”. Following is a brief description of the more common types of Amyloid (Table 2)
 
Alternative Classifications
An older, clinical, method of classification refers to amyloidosis as systemic or localized
Systemic amyloidosis affects more than one body organ or system (AL, AA, and Aβ2 M).
Localized amyloidosis affects only one body organ or tissue type Aβ, IAPP, atrial natriuretic factor in isolated atrial amyloidosis, and calcitonin in medullary carcinoma of the thyroid.
Another classification is primary or secondary.
Primary amyloidosis arises from a disease with disordered immune cell function such as multiple myeloma and other immunocyte dyscrasias.
Secondary (reactive) amyloidoses are those occurring as a complication of some other chronic inflammatory or tissue destructive disease. Examples are reactive systemic amyloidosis and secondary cutaneous amyloidosis.
 
Symptoms
There are numerous symptoms, which are associated with this disease. The most common ones have to do with the heart, such as heart failure, arrhythmia and an irregular heartbeat. Also, the respiratory tract can be affected and cause hemoptysis. Usually, the spleen enlarges and sometimes ruptures. The gastrointestinal tract is usually affected and causes vomiting, hemorrhaging and diarrhea. Amyloidosis can also affect the body's motor functions and cause polyneuropathy.
When the amyloid fibrils and oligomers get to the skin, they can cause skin lesions and petechiae. One of the most well-known symptoms is macroglossia (Figs 14A and B).94
Table 2   Amyloid classification
Abbreviation
Amyloid type/gene
Description
AL
Amyloid light chain
AL amyloidosis / Multiple myeloma. Contains immunoglobulin light chains (λ, κ) derived from plasma cells
AA
SAA
AA amyloidosis
β amyloid / APP
Found in Alzheimer's disease brain lesions
ATTR
Transthyretin
A mutant form of a normal serum protein that is deposited in the genetically determined familial amyloid polyneuropathies. TTR is also deposited in the heart in senile systemic amyloidosis. Also found in leptomeningeal amyloidosis
Aβ2M
β2 microglobulin
Not to be confused with Aβ, β2M is a normal serum protein, part of major histocompatibility complex (MHC) class 1 molecules. Hemodialysis associated amyloidosis
AIAPP
Amylin
Found in the pancreas of patients with type 2 diabetes
APrP
Prion protein
In prion diseases, misfolded prion proteins deposit in tissues and resemble amyloid proteins. Some examples are Creutzfeldt-Jakob disease (humans), BSE or “mad cow disease” (cattle), and scrapie (sheep and goats)
AGel
GSN
Finnish type amyloidosis
ACys
CST3
Cerebral amyloid angiopathy, Icelandic type
AApoA1
APOA1
Familial visceral amyloidosis
AFib
FGA
Familial visceral amyloidosis
ALys
LYZ
Familial visceral amyloidosis
?
OSMR
Primary cutaneous amyloidosis
ABri ADan
ITM2B
Cerebral amyloid angiopathy, British type Danish type
APro
Prolactin
Prolactinoma
AKer
Keratoepithelin
Familial corneal amyloidosis
AANF
Atrial natriuretic factor
Senile amyloid of atria of heart
ACal
Calcitonin
Medullary carcinoma of the thyroid
95
 
Diagnosis
If diagnosis of amyloidosis is suspected, a tissue sample of abdominal wall fat, the rectum or a salivary gland, can be examined in biopsy for evidence of characteristic amyloid deposits.
The tissue is treated with various stains. The most useful stain in the diagnosis of amyloid is Congo red, which, combined with polarized light, makes the amyloid proteins appear apple-green on microscopy. Alternatively, thioflavin T stain may be used. An abdominal wall fat biopsy is not completely sensitive and, sometimes, biopsy of an involved organ (such as the kidney) is required to achieve a diagnosis.
The nature of the amyloid protein can be determined by various ways: the detection of abnormal proteins in the bloodstream (on protein electrophoresis or light chain determination), binding of particular antibodies to the amyloid found in the tissue, or extraction of the protein and identification of its individual amino acids. The diagnosis of AL amyloidosis should be considered in patients with unexplained proteinuria, cardiomyopathy, neuropathy or hepatomegaly and in patients with multiple myeloma that has atypical manifestations.
The diagnosis of AL amyloidosis requires:
  • Demonstration of amyloid in tissue
  • Demonstration of a plasma cell dyscrasia.
Tissue amyloid deposits demonstrate apple-green birefringence when stained with Congo red and viewed under polarizing microscopy. Fine-needle aspiration of abdominal fat is a simple procedure that is positive for amyloid deposits in more than 70% of patients with AL amyloidosis.
Other tissues that allow for relatively noninvasive biopsy procedures are the minor salivary glands, gingiva, rectum, and skin. However, obtaining tissue from an affected organ may be necessary to establish the diagnosis of amyloidosis.
Once a tissue diagnosis of amyloidosis has been established, confirmation of AL disease requires demonstration of a plasma cell dyscrasia by a bone marrow biopsy showing predominance of λ or κ producing plasma cells or by the presence of a monoclonal light chain in the serum or urine.
Figs 14A and B: Macroglossia where the tongue is larger than normal
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Immunofixation electrophoresis should be performed on the serum and urine because, in contrast to multiple myeloma, the concentration of the monoclonal light chain often is too low to be detected by simple protein electrophoresis.
The recently introduced serum free light chain (FLC) assay, a nephelometric immunoassay, has a sensitivity for circulating free light chains that is reportedly more than tenfold that of immunofixation electrophoresis. Because the FLC assay is quantitative, it has utility not only in diagnosis, but also in following disease progression or response to treatment, as is discussed later. The normal concentrations of serum free light chains are 3.3–19.4 mg/L for κ isotype and 5.7–26.3 mg/L for λ isotype. The normal κ : λ ratio is 0.26–1.65.
Because free light chains undergo glomerular filtration, the ratio, rather than the absolute level, is the relevant measurement in individuals with renal impairment. A κ : λ ratio of less than 0.26 strongly suggests the presence of a population of plasma cells that are producing clonal λ free light chains, whereas a ratio more than 1.65 suggests production of clonal κ free light chains.
The diagnostic utility of the FLC is not firmly established but is under evaluation. In a study of 110 patients with a diagnosis of AL amyloidosis, serum immunofixation was positive in 69%, urine immunofixation was positive in 83%, and the κ : λ ratio by the FLC assay was abnormal in 91%. The combination of an abnormal κ : λ ratio and a positive serum immunofixation identified 99% of patients with AL amyloidosis. Another study of 169 patients with AL amyloidosis found that an abnormal free κ : λ ratio had greater specificity and predictive value than absolute levels of free light chains in patients with κ clonal disease.
Even if a monoclonal Ig light chain is identified in the serum or the urine, a bone marrow biopsy is mandatory to assess the plasma cell burden and exclude multiple myeloma and other, less common disorders that can be associated with AL amyloidosis, such as WM. It is important to recognize that the presence of a monoclonal band on serum immunofixation may be seen as an apparently incidental finding in 5–10% of patients who are older than 70 years, i.e. “monoclonal gammopathy of uncertain significance”.
Monoclonal gammopathy of undetermined significance : The serum FLC assay often is normal in such cases. Because of the high incidence of MGUS in elderly individuals, further testing should be done to exclude familial or senile forms of amyloidosis if the clinical picture is at all atypical for AL disease. Such testing includes immunohistochemistry, immunofluorescence or immunogold electron microscopy of amyloid deposits to identify the amyloidogenic protein or genetic testing to rule out familial forms of amyloidosis. Some laboratories measure total urine protein concentration and then decide how much to concentrate. Some others concentrate all samples 50–100x before applying them to the gel, but more and more laboratories are running urine samples neat.

Methods for ElectrophoresisCHAPTER 4

 
PRINCIPLE
Electrophoresis relies on the ability to separate out molecules on the basis of the electrical charge they possess. This is achieved by applying an electrical field induced between positive (anode) and negative (cathode) electrodes. Serum protein electrophoresis (SPE) is a screening test that measures the major blood proteins by separating them into five distinct fractions: albumin, α1, α2, β, and γ proteins. Serum proteins are separated in an electric field according to their size, shape, and electric charge. There is some difference in method, but basically the sample is placed in a special medium (e.g. a gel), and an electric current is applied to the gel. The protein particles move through the gel according to the strength of their electrical charges, forming bands or zones. The proteins are visualized by staining with acid blue, and the intensity of staining is quantitated by densitometry.
First, the hydrated support material (agarose gel or previously wetted cellulose acetate) is placed into the electrophoresis chamber. Care must be taken that excess buffer is removed from the support surface, and that bubbles are not present. The support is put into contact with buffer previously placed in the electrode chambers. Sample is applied to the support, and by using either constant voltage or constant current, electrophoresis is conducted for a determined length of time. The support is then removed from the electrophoresis cell and rapidly dried of placed in a fixative to prevent diffusion of sample components. Fixation is followed by treatment with a staining reagent to locate and reveal the individual protein zones. After excess dye is washed out, the support either is dried immediately or is first placed in a clearing agent. The result of electrophoresis of serum proteins is an electrophoretogram in which five major zones of proteins or protein groups can be seen, a typical electrophoretogram of a normal serum is illustrated in (Figures 1A and B).98
Fig. 1A: Typical normal pattern for serum protein electrophoresis.
Fig. 1B: The normal serum consequent “electrophoretic patterns” showing the locations of more commonly proteins
The modifications of each protein and a group of proteins must always be interpreted taking into consideration the synthesis, the distribution, the catabolism and therefore the physiopathological processes, which can modify one or more of these metabolic moments. Figure 1B shows the normal serum consequent “electrophoretic patterns” showing the locations of some of the more commonly known proteins.
The movement of ions within electrophoretic apparatus results in two currents, where negative ions (anions) flow from the cathode to anode, and positive ions (cations) move from anode to cathode. This results in an overall net movement of electrons from the cathode to anode, and drives the molecules carrying a particular charge in a given direction through a supporting matrix (e.g. polyacrylamide gels, detailed below). When a mixture of charged biomolecules is placed in an electrical field with defined field strength (E), they are carried towards the electrode of opposite charge. The secret underlying electrophoretic separation in a given experimental system is the fact that different biomolecules have varying physical characteristics (principally net charge, mass and shape) and a driving force called the Lorentz force (force on a point due to electric field) causes them to move at different rates through a given matrix. A given biomolecule in a mixture will have a net charge (q) that facilitates its movement in an electrical field (E). The velocity 99(v) at which a biomolecule will move in an electrical field increases with its net charge and the strength of the electrical field. However, the freedom of movement of biomolecules is restricted due to forces of friction (frictional coefficient (f), thus, decreasing their velocity through the electrical field. The magnitude of the frictional coefficient depends on factors, such as the mass and shape of the biomolecule and physical characteristics of the supporting matrix, including viscosity and porosity.
In a given electrophoretic system, an electrical field is established by applying a voltage (V) to a pair of electrodes (anode and cathode) separated by a particular distance (d). The electrical field strength (E) determines the ability and efficiency of a particular experimental system to separate charged biomolecules.
Electrophoretic mobility (μ) of a sample depends on the velocity (v) and electrical field strength (E), and biomolecules will migrate based on the ratio of net charge (q) to frictional coefficient (f).
In summary, the separation of biomolecules by electrophoresis depends on the experimental system in which they are being separated and the individual properties of biomolecules, namely net charge, mass and shape. Each of the principles outlined above are summarized by the following important equations:
 
WHY do WE PERFORM ELECTROPHORESIS?
Protein electrophoresis may be ordered to help in diagnosis of a disease, or it may be ordered to monitor treatment. When used to help in diagnosis, it may be ordered as a follow-up to abnormal findings on other laboratory tests or as an initial test in evaluating a person's symptoms. Once a disease or condition has been diagnosed, electrophoresis may be ordered at regular intervals to monitor the course of the disease and the effectiveness of treatment.
The primary reason for performing SPE is to discover a paraprotein or B-cell dyscrasia. An irregularity in the γ region can be due to a small monoclonal band, free light chains, or oligoclonal IgG. Other findings of clinical significance include increased α1 and α2 globulins indicative of an acute phase response, a decrease in α1 globulins suggestive of α1 antitrypsin (A1AT) deficiency (that can be followed up with phenotyping to check for a clinically significant A1AT variant), an increase in the β1 region suggestive of increased transferrin and iron deficiency, a polyclonal increase in γ globulins indicative of infection or of liver disease.
The main reason for performing urine protein electrophoresis is to find a light chain myeloma producing an excess of free light chains (Bence Jones protein), an important part of a myeloma screen. A band in the urine protein electropherogram may also result from an intact monoclonal immunoglobulin, especially if the patient has poor renal function. Immunofixation is important in defining the nature of the band and in 100distinguishing between Bence Jones protein and an intact monoclonal protein originating from the serum. From the urine electropherogram, we can also tell, if the proteinuria is of glomerular origin with a predominance of albumin, or if it has tubular components with excretion of smaller molecular weight proteins, such as retinol binding protein and α1 microglobulin. Fragmented albumin in urine is occasionally seen but is of unknown significance.
Historically, urine has been concentrated by either removal of water from the specimen leaving the proteins in higher concentration, or by centrifugation whereby the proteins are spun away from the majority of the water. Demonstration of the protein components of urine from concentrated specimens was originally performed on cellulose acetate and later on agarose and high resolution agarose gel. The use of capillary electrophoresis for urine analysis has not been accomplished to date by instrument manufacturers, such as Beckman or Sebia, although Sebia has promoted a method, which involves dialysis followed by a centrifugation step. An alternative urine protein method using capillary electrophoresis has been published. Some examples of when an electrophoresis test may be ordered are listed below.
  • Serum electrophoresis may be ordered:
    • As a follow-up to abnormal findings on other laboratory tests, such as total protein and/or albumin level, elevated urine protein levels, elevated calcium levels, or low- white or red blood cell counts
    • When symptoms suggest an inflammatory condition, an autoimmune disease, an acute or chronic infection, a kidney or liver disorder, or a protein losing condition
    • When a doctor is investigating symptoms that suggest multiple myeloma, such as bone pain, anemia, fatigue, unexplained fractures, or recurrent infections, to look for the presence of a characteristic band (monoclonal immunoglobulin) in the β or γ region; if a sharp band is seen, its identity as a monoclonal immunoglobulin is typically confirmed by immunofixation electrophoresis (IFE)
    • To monitor treatment of multiple myeloma to see if the monoclonal band is reduced in quantity or disappears completely with treatment.
  • Urine protein electrophoresis may be ordered:
    • When protein is present in urine in higher than normal amounts to determine the source of the abnormally high protein, it may be used to determine whether the protein is escaping from the blood plasma (suggesting compromised kidney function) or is an abnormal protein coming from a different source (such as a plasma cell cancer like multiple myeloma).
    • When multiple myeloma is suspected to determine whether any of the monoclonal immunoglobulins or fragments of monoclonal immunoglobulin are escaping into the urine (if a sharp band suggestive of a monoclonal protein is observed), its identity is typically confirmed by IFE.
  • Cerebrospinal fluid (CSF) protein electrophoresis may be ordered:
    • To search for the characteristic banding seen in multiple sclerosis ; the presence of multiple distinct bands in the CSF (that are not also 101present in serum) are referred to as oligoclonal bands. Most people with multiple sclerosis, as well as some other inflammatory conditions of the brain have such oligoclonal bands
    • To evaluate people having headaches or other neurologic symptoms to look for proteins suggestive of inflammation or infection.
  • Immunofixation electrophoresis may be ordered:
    • When an abnormal band suggestive of a monoclonal immunoglobulin is seen on either a serum or a urine electrophoresis pattern.
 
SAMPLE COLLECTION
 
Serum Proteins
Fresh serum sample is the preferred specimen. The use of plasma should be avoided, as fibrinogen will appear as a distinct narrow band between the β and γ fractions (no anticoagulant should be used). The sample for the SPE test is obtained by venipuncture (Fig. 2). It is usually not necessary for the patient to restrict food or fluids before the test. A 12-hour fast is requested before drawing blood for lipoprotein testing.
 
Urinary Proteins
The urine protein electrophoresis test requires either an early morning urine sample or a 24-hour urine sample according to the physician's request. Protein electrophoresis is performed on urine samples to classify disorders, which cause protein loss via the kidneys. Hemoglobin and myoglobin are found in the urine of trauma and burn victims, and in patients with infection or Hemolysis. Protein electrophoresis of urine is most often performed in order to detect the presence of light chain fragments of immunoglobulins. These protein fragments are sufficiently small to filter through the kidneys and are excreted in the urine. They are called Bence Jones proteins, and are found in patients who have multiple myeloma, a malignant proliferation of antibody producing cells. Bence Jones proteins may also be found in other variants of multiple myeloma, such as light chain disease, and in patients with systemic autoimmune diseases that result from degradation of immune complexes; Urine without preservative any may be used if concentrated up to 300 times, depending on original protein concentration.
Fig. 2: Only a trained person is able to perform the blood collection procedure. The sample should be collected from the most suitable vein and clean the site
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Cerebrospinal Fluid Proteins
Cerebrospinal fluid is collected by lumbar puncture performed in a hospital setting. Because of risks associated with the procedure, the patient must sign a consent form. Any factors that might affect test results, such as whether the patient is taking any medications, should be noted. An increase in total protein concentration in the CSF is often found in bacterial and fungal meningitis and with central nervous system (CNS) tumors. The main use of CSF protein electrophoresis testing is in the diagnosis of multiple sclerosis, CSF may be used if concentrated approximately 100 times.
In performing a lumbar puncture, first the patient is usually placed in a left (or right) lateral position with his/her neck bent in full flexion and knees bent in full flexion up to his/her chest, approximating a fetal position as much as possible. It is also possible to have the patient sit on a stool and bend his/her head and shoulders forward. The area around the lower back is prepared using aseptic technique. Once the appropriate location is palpated, local anesthetic is infiltrated under the skin and then injected along the intended path of the spinal needle. A spinal needle is inserted between the lumbar vertebrae L3/L4 or L4/L5 and pushed in until there is a “give” that indicates the needle is past the ligamentum flavum (Fig. 3). The needle is again pushed until there is a second “give” that indicates the needle is now past the dura mater. Since the arachnoid membrane and the dura mater exist in flush contact with one another in the living person's spine (due to fluid pressure from CSF in the subarachnoid space pushing the arachnoid membrane out towards the dura), once the needle has pierced the dura mater it has also traversed the thinner arachnoid membrane and is now in the subarachnoid space. The stylet from the spinal needle is then withdrawn and drops of CSF are collected. The opening pressure of the CSF may be taken during this collection by using a simple column manometer. The procedure is ended by withdrawing the needle while placing pressure on the puncture site. In the past, the patient would often be asked to lie on his/her back for at least 6 hours and be monitored for signs of neurological problems, though there is no scientific evidence that this provides any benefit.
Fig. 3: A patient undergoes a lumbar puncture
103
The technique described is almost identical to that used in spinal anesthesia, except that spinal anesthesia is more often done with the patient in a seated position.
The upright seated position is advantageous in that there is less distortion of spinal anatomy, which allows for easier withdrawal of fluid. It is preferred by some practitioners when a lumbar puncture is performed on an obese patient where having them lie on their side would cause a scoliosis and unreliable anatomical landmarks. On the other hand, opening pressures are notoriously unreliable when measured on a seated patient, and therefore, the left or right lateral (lying down) position is preferred, if an opening pressure needs to be measured.
Patient anxiety during the procedure can lead to increased CSF pressure, especially if the person holds their breath, tenses their muscles or flexes their knees too tightly against their chest. Diagnostic analysis of changes in fluid pressure during lumbar puncture procedures requires attention both to the patient's condition during the procedure and to their medical history.
 
Bone Marrow Sample
In some situations the hematologist may recommend that a sample of bone marrow is taken (biopsy) to be examined under a microscope.
The sample is usually taken from the back of the hip bone (pelvis). An injection of local anesthetic to numb the area will be given. The doctor will then pass a needle through the skin into the bone and draw a small sample of liquid marrow into a syringe (bone marrow aspirate). After this, the doctor will take a small core of marrow from the bone [a trephine biopsy (Figs 4A to C]. Both samples will be looked at later under a microscope.
 
Precautions
Some other diagnostic tests or prescription medications can affect the results of protein electrophoresis tests. The administration of a contrast dye used in some other tests may falsely elevate apparent protein levels. Drugs that can alter results include aspirin, bicarbonates, chlorpromazine (Thorazine), corticosteroids, isoniazid (INH), and neomycin (Mycifradin). The total serum protein concentration may also be affected by changes in the patient's posture or by the use of a tourniquet during the drawing of blood. Studies show that differences are seen in pregnant females and in women on oral contraceptives. In addition, age has some effect on normal levels, and the cord blood has decreased total protein, albumin, α2 and β fractions.
Protein is less concentrated in urine and CSF than in blood. Urinary and CSF proteins must be concentrated before analysis, and the added sample handling can lead to contamination and erroneous results. In the collection of a CSF specimen, it is important, that the sample not be contaminated with blood proteins that would invalidate the CSF protein measurements.104
Figs 4A to C: A. bone marrow sample aspiration; B. A needle used for bone marrow aspiration with remo stylet; C. The oncontrol bone marrow biopsy and aspiration system
 
Description
Proteins are biologically important organic molecules or polymers of amino acids that contain the elements, such as carbon, hydrogen, nitrogen, and oxygen. Some proteins may also contain sulfur, phosphorus, iron, iodine, selenium, or other trace elements. There are 22 amino acids commonly found in all proteins. The human body is capable of producing 14 of these amino acids, and the remaining eight so-called essential amino acids must be obtained from food. Proteins are found in muscles, blood, skin, hair, nails, and the internal organs and tissues. Enzymes, hemoglobin, and antibodies are proteins, as are many hormones. Protein mixtures can be fractionated into individual component proteins by a variety of techniques, including precipitation, chromatography, ultracentrifugation, or electrophoresis.
A SPE test is used to determine the percentage of each protein in the blood by separating them into five distinct classes: albumin, α1 globulin, α2 globulin, β globulin, and γ globulins (immunoglobulins). High-resolution 105protein electrophoresis uses a higher current to separate the major proteins comprising the α1 globulin, α2 globulin, and β globulin fractions. This procedure produces nine or more bands, including α1 antitrypsin, α2 macroglobulin, haptoglobulin, transferrin, and complement proteins.
In addition to standard protein electrophoresis, the IFE test may be used to assess the blood levels of specific immunoglobulins. An IFE test is usually ordered if a SPE test shows an unusually high amount of protein in the γ globulin fraction. The IFE tests determine whether the increase in the γ globulin fraction is caused by excess immunoglobulins (antibodies), and whether it is polyclonal or monoclonal in nature. Polyclonal increases are caused by infections, allergies, and inflammatory diseases, while monoclonal increases are caused by malignant or benign proliferations of the antibody producing cells (plasma cells).
 
Storage and Stability
If storage is necessary, samples may be stored covered at 2–8°C for 48 hours. CSF and urine specimens may be used after proper concentration with a concentrator.
 
PROTEIN STAINS FOR ELECTROPHORESIS PROCEDURES
 
Protein Gel Stains
Once protein bands have been separated by electrophoresis, they can be visualized using different methods of in-gel detection. Over the past several decades, demands for improved sensitivity for small sample sizes and compatibility with downstream applications and detection instrumentation have driven the development of several basic staining methods over the years. Each method has particular advantages and disadvantages, and a number of specific formulations of each type of method provide optimal performance for various situations. This section discusses the general principles of protein gel staining and describes several types of staining methods.
 
General Principles of Gel Staining
  • Coomassie dye stains
  • Amido black (naphthol blue black) stains
  • Bromophenol blue stains
  • Nigrosin stains
  • Silver stains
  • Zinc stains
  • Ponceau S stains
  • Fluorescent dye stains
  • Functional group specific stains.
 
General Principles of Gel Staining
The first step after performing denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) is to disassemble the gel 106cassette and place the thin (1 mm) polyacrylamide gel in a tray filled with water or buffer. The electrophoresed proteins exist as concentrated “bands” embedded within each lane of the porous polyacrylamide gel matrix. Typically, the proteins are still bound to anionic SDS detergent, and the entire gel matrix is saturated in a particular buffer.
To make the proteins visible, a protein-specific, dye-binding, or color, producing chemical reaction must be performed on the proteins within the gel. Depending on the particular chemistry of the stain, various steps are necessary to hold the proteins in the matrix and to facilitate the necessary chemical reaction. All steps are done in solution, i.e. with the gel suspended in a tray filled with one liquid reagent or another.
Given the common constraints of this format, most staining methods involve some version of the same general incubation steps:
  • A water wash to remove electrophoresis buffers from the gel matrix
  • An acid or alcohol wash to condition or fix the gel to limit diffusion of protein bands from the matrix
  • Treatment with the stain reagent to allow the dye or chemical to diffuse into the gel and bind (or react with) the proteins
  • Destaining to remove excess dye from the background gel matrix.
Depending on the particular staining method, two or more of these functions can be accomplished with one step. For example, a dye reagent, which is formulated in an acidic buffer can effectively fix and stain in one step. Conversely, certain functions require several steps. For example, silver staining requires both a stain reagent step and a developer step to produce the colored reaction product.
 
Coomassie Dye Stains
Coomassie dye stain is the most popular reagent for staining protein bands in electrophoretic gels. In acidic buffer conditions, Coomassie dye binds to basic and hydrophobic residues of proteins, changing from dull reddish brown to intense blue. As with all staining methods, Coomassie dye reagents detect some proteins better than others based on their chemistry of action and differences in protein composition. For most proteins, however, Coomassie dye reagents detect as few as 10 nanograms per band in a mini gel. Several Coomassie stain reagent recipes exist in the literature and use either the G 250 (colloidal) or R 250 form of the dye. Colloidal Coomassie can be formulated to effectively stain proteins within 1 hour and require only water (no methanol or acetic acid) for destaining.
In acidic buffer conditions, Coomassie dye binds to basic and hydrophobic residues of proteins, changing from dull reddish brown to intense blue. As with all staining methods, Coomassie dye reagents detect some proteins better than others based on their chemistry of action and differences in protein composition. Thus, coomassie dye reagents can detect as few as 8–10 ng per band for some proteins and 25 ng per band for most proteins.
Coomassie dye staining is especially convenient, because it involves a single, ready-to-use reagent and does not permanently chemically modifies the target proteins. An initial water wash step is necessary to remove residual 107SDS, which interferes with dye binding.
Fig. 5: Amido black (naphthol blue black) structure
Then stain reagent is added, usually for about 1 hour; finally, a water or simple methanol-acetic acid destaining step is used to wash away excess nonbound dye from the gel matrix. Because no chemical modification occurs, excised protein bands can be completely destained and the proteins recovered for analysis by mass spectrometry (MS) or sequencing.
 
Amido Black (Naphthol Blue Black) Stains
Amido black [naphthol blue black (Fig. 5)] stain is a dye that is highly sensitive to the protein compounds found in blood. It is an acid disazo dye that is made by coupling one mole each of p-nitroaniline and aniline to one mole of tetra-azotized 8-amino-1-naphthol 3,6-disulfonic acid (H acid). The p-nitroaniline coupling is accomplished at a pH of 5–6 while the aniline is coupled at a pH of 8–10. Amido black is an extremely effective protein stain used to enhance faint or nearly invisible bloodstains to a deep blue black color. It will not stain latent prints that are not bloodstained and therefore must be used after other latent print processes have been performed. It is also important that any biological evidence, such as semen, urine, or blood have been collected before use. Amido black can be used on most porous and nonporous surfaces. Amido black can be either methanol or water based. The reaction product is a strong blue/black color, and it is best suited for use on light colored surfaces permitting good visual contrast. Amido black solution is now supplied in a pump spray eliminating any advance preparation prior to use at the crime scene. When stored at room temperatures, the solution offers a shelf life of up to 1 year. While amido black is nonflammable, it is corrosive and will stain many surfaces. Detection sensitivity is approximately 20% that of coomassie blue R.
 
Bromophenol Blue Stains
Bromophenol blue stain (Fig. 6) is used as a color marker to monitor the process of agarose gel electrophoresis and polyacrylamide gel electrophoresis. Since bromophenol blue carries a slight negative charge at moderate pH, it will migrate in the same direction as DNA or protein in a gel; however, the rate at which it migrates varies according to gel density and buffer composition, but in a typical 1% agarose gel in TAE buffer or TBE 108buffer, bromophenol blue migrates at the same rate as a DNA fragment of approximately 500 base pairs.
Fig. 6: Bromophenol blue structure
Xylene cyanol and o range G may also be used for this purpose. Bromophenol blue is also used as a dye, at neutral pH, the dye absorbs red light most strongly and transmits blue light. Solutions of the dye therefore are blue. At low pH, the dye absorbs ultraviolet and blue light most strongly and appears yellow in solution. In solution at pH 3.6 (in the middle of the transition range of this pH indicator) obtained by dissolution in water without any pH adjustment, bromophenol blue has a characteristic green-red color. This phenomenon is called dichromatic color. Bromophenol blue is the substance with the highest known value of Kreft's dichromaticity index. This means that it has the largest change in color hue, when the thickness or concentration of observed sample increases or decreases.
 
Nigrosin Stains
Nigrosin stain is the forgotten protein stain. It is a mixture of synthetic black dyes made by heating a mixture of nitrobenzene, aniline and aniline hydrochloride in the presence of a copper or iron catalyst. Its main industrial uses are as a colorant for lacquers and varnishes and in marker pen inks. Sulfonation of nigrosin yields a water soluble anionic dye.
In biology, nigrosin is used for negative staining of bacteria. The shapes and sizes of the organisms are seen as color-free outlines against the dark background. An advantage of using this method, rather than regular positive stains like methylene blue or carbol fuchsin, is that prior fixation by heat or alcohol is not needed, so the organisms are seen in more life like shapes. Furthermore, negative staining with nigrosin can reveal some microorganisms that cannot be stained by regular methods.
 
Silver Stains
Another popular method for detecting protein bands within a gel is silver staining that deposits metallic silver onto the surface of a gel at the location of protein bands. Commercial silver stain kits are exceptionally robust and easy to use. It is the most sensitive colorimetric method for detecting total protein.109
Fig. 7: Silver staining of the gel was performed
The technique involves the deposition of metallic silver onto the surface of a gel at the location of protein bands. Silver ions (from silver nitrate in the stain reagent) interact and bind with certain protein functional groups. Strongest interactions occur with carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryls (Cys), and amines (Lys). Various sensitizer and enhancer reagents are essential for controlling the specificity and efficiency of silver ion binding to proteins and effective conversion (development) of the bound silver to metallic silver. The development process is essentially the same as for photographic film. Silver ions are reduced to metallic silver, resulting in brown black color (Fig. 7).
Silver staining protocols require several steps, which are affected by reagent quality as well an incubation times and thickness of the gel. An advantage of commercially available silver staining kits is that the formulations and protocols are optimized and consistently manufactured, helping minimize the effects of minor differences in day-to-day use. Kits with optimized protocols are robust and easy to use, detecting less than 0.5 nanograms of protein in typical gels.
Silver stains use either glutaraldehyde or formaldehyde as the enhancer. These reagents can cause chemical crosslinking of the proteins in the gel matrix, limiting compatible with destaining and elution methods for analysis by MS. Therefore, optimization of sensitivity versus protein recoverability is critical when silver staining as part of an MS workflow.
Silver stain formulations can be made such that protein bands stain black, blue brown, red or yellow, depending on their charge and other characteristics. This is particularly useful for differentiating overlapping spots on two-dimensional (2D) gels.
 
Zinc Stains
The zinc stain is unique and is unlike all other staining methods. Instead of staining the proteins, this procedure stains all areas of the polyacrylamide gel in which there are no proteins. Zinc ions complex with imidazole, precipitates 110in the gel matrix except where SDS saturated protein occur.
Fig. 8: Zinc staining of the gel was performed
The milky white precipitate renders the background opaque while the protein bands remain clear. The process is short, about 15 minutes, and the gel can be photographed by viewing the gel over a dark background. Zinc staining is as sensitive as typical silver stains (detects < 1 ng of protein), and there are no fixation steps. Furthermore, the stain is easily removed, making this method compatible with MS or Western blotting. Figure 8 shows proteins were electrophoresed that stained with the pierce zinc reversible stain kit, and then photographed with the gel placed over a dark blue background; most proteins are easily visible at 0.25 ng.
 
Ponceau S Stains
Ponceau S (Fig. 9) stain is a sodium salt of a diazo dye that may be used to prepare a stain for rapid reversible detection of protein bands on nitrocellulose or polyvinylidene difluoride (PVDF) membranes (Western blotting), as well as on cellulose acetate membranes. Ponceau S stain is a ready-to-use, designed for rapid (5 minutes) staining of protein bands. It is easily reversed with water washes, facilitating subsequent immunological detection. A Ponceau S stain is useful, because it does not appear to have a deleterious effect on the sequencing of blotted polypeptides and is therefore one method of choice for locating polypeptides on Western blots for blot sequencing. It is also easily reversed with water washes, facilitating subsequent immunological detection. The stain can be completely removed from the protein bands by continued washing. Common stain formulations include 0.1% (w/v), Ponceau S in 5% acetic acid, or 2% (w/v) Ponceau S in 30% trichloroacetic acid (TCA) and 30% sulfosalicylic acid.
The stain may be stored as packaged or in a tightly-closed staining dish at 15–30°C. The unopened stain is stable until the expiration date on the bottle. The stain should be a homogeneous mixture free of precipitate.111
Fig. 9: Ponceau S structure
 
Fluorescent Dye Stains
In recent years, the demand for fluorescent stains has increased with the improvements and popularity of fluorescence imaging technique. Fluorescent stains are now available with excitation and emission maxima corresponding to the common filter sets and laser settings of most fluorescent imagers. Fluorescent imagers and fluorescent applications have resulted in greater demand for fluorescent stains beyond the traditional ethidium bromide stain for nucleic acids. A number of total protein fluorescent stains have been introduced in recent years. Newer fluorescent total protein stains provide exceptional fluorescent staining performance with a fast and easy procedure. The most useful ones are those whose excitation and emission maxima corresponding to common filter sets and laser settings of popular fluorescent imagers. Most fluorescent stains involve simply dye binding mechanisms rather than chemical reactions, which alter protein functional groups. Therefore, most of them are compatible with destaining and protein recovery methods for downstream analysis by MS. Accordingly, they are frequently used in both one-dimensional (1D) and 2D applications.
 
Functional Group-specific Stains
Sometimes, it is desirable to detect a subset of proteins rather than all of the proteins in a gel. Glycoproteins and phosphoproteins are categories of proteins that are classified on the basis of a particular type of chemical moiety (i.e. polysaccharides and phosphate groups, respectively). When a dye-binding or color-producing chemistry can be designed to detect one of these functional groups, it can be used as the basis for a specific gel stain.112
Proteins that have been post-translationally modified by glycosylation can be detected by a procedure, which involves chemical activation of the carbohydrate into a reactive group. The method works by fixing the proteins in the gel and then oxidizing the sugar residues with sodium metaperiodate. The resulting aldehyde groups can then be reacted with an amine containing dye. In older literature, this method is known as the periodate acid Schiff (PAS) technique. A subsequent reduction step stabilizes the dye protein bond. Both colorimetric and fluorescent dyes have been used and glycoprotein stain kits are available commercially.
Another post-translational modification that can be detected is phosphorylation. When significant amounts of phosphorylated proteins are present, the phosphate groups can be cleaved from phosphoserine and phosphothreonine residues and precipitated with calcium. Then the resulting precipitate can be detected with molybdate and methyl green. When this method is used, the same gel can be stained for total protein content with a Coomassie dye stain.
Finally, several traditional and innovative chemistries exist for staining specific classes of proteins in polyacrylamide gels. These include commercial stain kits to detect glycoproteins or phosphoproteins.
Stains used to locate and reveal the separated protein fractions of the sample differ in accordance with the type of application and the personal choice of the analyst. The amount of dye bound by each protein zone is related to the amount of protein present in the zone but is affected by many other factors, such as the type of protein present and the degree of denaturation caused by the fixing. Recently, silver nitrate has become popular, because of its sensitivity for staining of proteins and polypeptides.
 
QUANTITATION OF PROTEIN ZONES
Customarily, results of electrophoretic separation are reported in terms of the percentage of each fraction present or in terms of absolute concentration when the concentration of total protein is known. Quantitation of the amount of dye in individual zones can be conveniently accomplished either by direct densitometry or by elution of dye from individual zones and by subsequent spectrophotometric measurement of eluted dye. In densitometry, the fixed and cleared electrophoretic medium is moved past the optical system of and instrument, called a densitometer; the location and intensity of zone are indicated as successive peaks on a recorder chart. In some instruments, the area under a peak is automatically integrated. The elution method involves cutting the support into separate zones and eluting the adsorbed dye in each with suitable solvents, such as basic buffers of alcoholic solutions (Table 1).
Tables 27 illustrate the position, morphological aspects, and the function of the proteins, which can be highlighted in area electrophoretic separation:113
Table 1   Stains for visualization of serum protein zones and wavelengths for quantitation by densitometry or elution spectrophotometry
Application
Stain
Nominal wavelength (nm)
Densitometry
Elution
Staining of serum proteins in general
Amido black (Naphthol blue black)
640
600
Bromophenol blue
600
595
Coomassie brilliant blue G 250 (brilliant blue G)
595
610
Coomassie brilliant blue R 250 (brilliant blue R)
560
580
Nigrosin
540
560
Ponceau S
520
565 (alkaline)
520 (neutral)
Staining of isoenzymes
Nitrotetrazolium blue (as the formazan)
570
_
Staining of lipoproteins
Sudan red 7B
540
_
Oil Red O
520
_
Sudan black B
600
_
Table 2   Albumin area (from the anode to the cathode)
Specific protein
Position and morphological aspect
Function
Prealbumin
Thin band
Transport
Albumin
Intense band
Transport and oncotic function
Table 3   Interalbumin α1 antitrypsin area (from anode to cathode)
Specific protein
Morphological aspect and/or position
Function
Apo A
Usually diffused sometimes at undulating band
HDL polyclonal component
α1 acid glycoprotein
For concentration > 200 mg/dL generates a diffused thin band
Acute phase
α1 antitrypsin genotype FF
Clear tape
Protease inhibitor
α1 antitrypsin genotype MM
Clear marked
Protease inhibitor
114
Table 4   Interalbumin α1 antitrypsin α3 macroarea (from anode to cathode)
Specific protein
Morphological aspect and/or position
Function
α1 antitrypsin genotype MS
Two tapes, one central and one toward α2 macro
Protease inhibitor
α1 antitrypsin genotype MZ
Two tapes, one central and one close to α2 macro
Protease inhibitor
α1 antitrypsin genotype SS
Decentralized tape toward α2 macro
Protease inhibitor
α1 antitrypsin genotype ZZ
Tape close to α2 macro
Protease inhibitor
Inter α-trypsin inhibitor
Weak bands with diffused surrounding areas located in the genotypes area α1 antitrypsin.
Protease inhibitor
Table 5   Inter α2 macrotransferrin area (from anode to cathode)
Specific protein
Morphological aspect and/or position
Function
α2 macroglobulin
In relation to the positions assumed by the haptoglobulin phenotypes may not be well delineated
Protease inhibitor and factors of coagulation, fibrinolysis and complement
Apo β
It can migrate anodically or cathodically to the transferrin. When it is visible it always has a morphology, which is undulating or with arched extremities
LDL/VLDL polyclones component
C3
For activation in vitro or in vivo it can be anodical to the transferrin
Activation of the C3 in the complement cascade
 
DENSITOMETRY
Densitometry has become the method of choice in most clinical laboratories. The following features should be sought when choosing a densitometer: (i) ability to scan electrophoresis supports with lengths of 25–150 mm, (ii) automatic gain control to prevent the response for the most intense peak or zone from going off scale, (iii) automatic zeroing capability for background correction (this feature allows the instrument to choose the lowest background point in the electropherogram for a baseline so that minor peaks are not lost or cut off), (iv) a variable wavelength control, either as a continuously variable monochromator or as variably selected interference filters to allow operation in the range of 400–700 nm, (v) variable slits form 0.1 × 2.0 to 2.0 × 10.0 mm (this feature allows the instrument to discriminate between individual narrow bands), and (vi) an integrating device.115
Table 6   Intertransferrin immunoglobulin area (from anode to cathode)
Specific protein
Morphological aspect and/or position
Function
Transferrin
It appears as a distinct band. Slow and quick variations are highlighted. The heterozygote shapes appear as two distinct and equally intense bands
The greatest protein-iron bond and transport
Cryoglobulins
From light to marked deposit sign. Sometimes, if present, a MC can migrate into a γ area (usually an IgM)
Cryoglobulinemia
C3
It appears as a distinct band
Anaphylotoxin C3a Opsonization C3b, Chemotaxis C3b, etc.
C4
Can be anodically found at C3 as thin band during inflammatory processes
Fourth component of the complement cascade
Fibrinogen
Can appear as a weak band located between C3 and the beginning of the γ area for an incomplete coagulation or for withdrawal of serum with issue of FDP from coagulation
Precursor of the fibrin
Table 7   Immunoglobulin area (from anode to cathode)
Specific protein
Morphological aspect and/or position
Function
Immunoglobulin
Long area diffused with anodic confines in the β area, and cathodic confines up to the retromigrations
Antibodies
C-reactive proteins
Light or distinct band in γ central area
Activator of the complement
Monoclonal components
Light or distinct bands more frequently in γ area but which can migrate from the α1 area to the post-γ area
Multiple myeloma?
Immunocomplexes
Light distinct bands, usually two in number in γ area
Cryoglobulinemia?
The simplest integrator provides zigzag integration, requiring the operator to count the number of sawteeth and to compute manually the area of the zones integrated. Microprocessor fitted models, however, rapidly and automatically compute areas of peaks and usually provide printouts. When automatic computation is desired, one should consider a densitometer 116capable of integrating up to 30 peaks rather than one limited to the five zones associated with conventional SPE. Such a choice allows the analyst to apply the instrument to those high-resolution electrophoretic is unique that fractionate serum proteins more completely. Other desirable features are an ultraviolet fight source and the ability to measure fluorescence. Additional features, not essential but helpful, are automatic indexing (a feature that automatically advances an electrophoretic strip containing multiple samples from one sample to the next), built-in diagnostics for instrument troubleshooting, and a choice of several scanning speeds.
The linearity of response of a densitometer may be tested by standards available from various instrument manufacturers. Standards have been designed with several separate zones of increasing density or with gradually increasing density. The first type causes the recorder pen to return to baseline between zones. By evaluating the behavior of peak tracing and baseline return, the operator can check the optical, mechanical, and electrical functions of the densitometer. Densities of zones in the standard are compared with their expected values. With modern cellulose acetate and agarose gel techniques, the use of very small sample size, as well as excellent transparency when strips are scanned, has substantially eliminated the shortcomings of densitometry observed with opaque cellulose paper strips. Nevertheless, problems persist, because of differences in affinity by individual proteins for stain and differences in size among protein zones.
After electrophoresis, a stained gel is passed through the optical system of a densitometer to create an electropherogram, a visual diagram or graph of the separated bands (Fig. 10). A densitometer is a special spectrophotometer that measures light transmitted through a solid sample, such as a cleared or transparent but stained gel. Using the optical density measurements, the densitometer represents the bands as peaks.
These peaks compose the graph or electropherogram and are printed on a recorder chart or computer display. Absorbance and/or fluorescence can be measured with densitometry. An integrator or microprocessor evaluates the area under each peak and reports, each as a percent of the total sample.
Fig. 10: Densitometer scan of serum protein electrophoresis
117
Fig. 11: A commercially available power supply
If the electrophoresis is for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration. If the electrophoresis is for separation of enzymes, the enzyme activity of each band is derived from this percent and the total enzyme activity.
 
POWER SUPPLY
Commercially available power supplies (Fig. 11) allow operation at either constant current or constant voltage. The flow of current through a medium that offers electrical resistance is associated with the production of heat:
where (E) is electromotive force in volts (V), (I) is current tin amperes (A), and (t) is time in seconds (s). Heat evolved during electrophoresis increases the conductance of the system (i.e. resistance decreases), as a result of the increase in thermal agitation of all dissolved ions. When a constant voltage power source is used, the resultant rise in current causes a progressive increase in the migration rates of proteins (Ohm's law, E = IR). The rate of evaporation of water from the support medium is also increase in resistance (R). These effects on the migration rate are best minimized by the use of a constant current power supply. In this case, a decrease in R due to heat also decreases V, and the current remains constant. As a result, the heat effect is decreased and the migration rate is kept relatively constant.
 
TYPES OF ELECTROPHORESIS
Electrophoresis is a term, which describes both a concept and technique or experimental system. There are many applications of electrophoresis, and this technique is still finding new applications in a wide range of scientific disciplines. A large number of variants of gel electrophoresis are used in bioanalytical analysis to allow separation and characterization of biomolecules.
 
Paper Electrophoresis
Paper electrophoresis [PE (Fig. 12)], widely used in clinical laboratories for the separation of serum proteins, has largely been replaced by cellulose 118acetate or agarose gel electrophoresis.
Fig. 12: Paper electrophoresis apparatus
Significant disadvantages in the use of PE include the long separation time (14–16 hours), excessive background (tailing), and decreased resolution. The advantages of paper as a support medium are high tensile strength, low cost, and ease of handling.
 
Agar Gel Electrophoresis
Agar gel electrophoresis (AGE) has been successfully applied to the analysis of serum proteins, hemoglobin variants, lactate dehydrogenase isoenzymes, lipoprotein fractions, and other substances. This medium parallels cellulose acetate in versatility, convenience, and applicability to routine clinical demands. However, agar contains at least two fractions: agaropectin and agarose. Agaropectin has acid sulfate and carboxylic acid groups and accounts for the considerable endosmosis and background staining that are observed on electropherograms made on unfractionated agar. Agarose that is essentially free on ionizable groups, exhibits little endosmosis and has replaced agar in routine clinical laboratory procedures. Occasionally, lots of commercially available agarose, however, contain some residual charged groups. Agarose gel has a low affinity for protein and has very little effect on migration rate. Because it is clear after drying, it permits excellent densitometric examination. Usually 0.5–1.0 g of agarose/dL of buffer provides a gel with the desired strength and with good migration properties. Unmodified serum or serum dissolved in warmed agarose is applied directly into a precut or precut or precast well. Although the latter technique is less convenient, it has the advantage that the agarose sample solution solidifies to become part of the agarose support. In contrast, the direct application of serum to a precut slot causes an uneven surface at the sample application points and an artifactual peak in densitometry. Another alternative uses a thin plastic template that has small slots corresponding to sample application points. The template is placed upon the agarose surface and 5 μL samples are introduced into each slot.119
 
Cellulose Acetate Electrophoresis
When the hydroxyl groups of cellulose are reacted with acetic anhydride, cellulose acetate is formed. This acetylated cellulose forms the raw material for cellulose acetate membranes. The membranes commercially available contain about 80% airspace in the form of pockets within the interlocking cellulose acetate fibers. The membranes as purchased are dry, opaque, brittle films can crack easily if not handled gently. When the film is placed in buffer, the airspaces fill with liquid, and the film becomes quite pliable. Characteristics of specific membranes vary with the extent of acetylation, as well as with the pore size and thickness of the membrane. Cellulose acetate that has been especially prepared to reduce electroendosmosis is also commercially available.
Serum samples (0.3–2.0 μL) are generally applied with a twin-wire applicator to cellulose acetate strips that have been presoaked in buffer. Cellulose acetate membranes may be made transparent (cleared) for densitometry by treatment with a solvent mixture containing first solvent that dissolved cellulose acetate, and a second solvent that promotes miscibility between the buffer and the first solvent. A typical clearing solution consists of 95 parts methanol and 5 parts glacial acetic acid. The cellulose acetate fibers partially dissolved by the action of the solvent coalesce, thus, eliminating the airspaces responsible for the original opacity.
Advantages of cellulose acetate electrophoresis (CAE) are the speed of separation (20 minutes to 1 hour) and the stability of the cleared membranes when stored for long periods.
 
Polyacrylamide Gel Electrophoresis
Protein electrophoresis on paper, cellulose acetate, or some agarose gels yields only five to seven zones owing to diffusion during the electrophoretic procedure. Polyacrylamide, or starch gel, electrophoresis commonly yields 20 or more fractions and is, therefore, widely used to study individual serum proteins, particularly genetic variants and isoenzymes.
The polyacrylamide (Fig. 13) gel electrophoresis (PAGE) technique employs layers of gel that bigger in compositions and pore size.
Fig. 13: Polyacrylamide structure
Because of 120the discontinuities of the electrophoretic matrix and the discoid shape of the separated zones of protein, this method is sometimes referred to as disk electrophoresis. This technique, which was introduced in 1964, minimizes band broadening due to diffusion.
Acrylamide is the material of choice for preparing electrophoretic gels to separate proteins by size. Acrylamide mixed with bisacrylamide forms a cross-linked polymer network when the polymerizing agent, ammonium persulfate (APS), is added. N,N,N,N’ tetramethylenediamine (TEMED) catalyzes the polymerization reaction by promoting the production of free radicals by APS.
The ratio of bisacrylamide to acrylamide as well as the total concentration of both components affect the pore size and rigidity of the final gel matrix. These, in turn, affect the range of protein sizes (molecular weights), which can be resolved.
The size of the pores created in the gel is inversely related to the amount of acrylamide used. A 7% polyacrylamide gel has larger pores than a 12% polyacrylamide gel. Gels with a low percentage of acrylamide are typically used to resolve large proteins, and high percentage gels are used to resolve small proteins. “Gradient gels” are specially prepared to have low percent acrylamide at the top (beginning of sample path) and high percent acrylamide at the bottom (end), enabling a broader range of protein sizes to be separated.
Electrophoresis gels are formulated in buffers that provide for conduction of an electrical current through the matrix. The solution is poured into the thin space between two glass or plastic plates of an assembly called a “cassette”. Once the gel polymerizes, the cassette is mounted (usually vertically) into an apparatus so that opposite edges (top and bottom) are placed in contact with buffer chambers containing cathode and anode electrodes, respectively. When proteins are added in wells at the top edge and current is applied, the proteins are drawn by the current through the matrix slab and separated by its sieving properties.
To obtain optimal resolution of proteins, a “stacking” gel is cast over the top of the “resolving” gel. The stacking gel has a lower concentration of acrylamide (e.g. 7% for larger pore size), lower pH (e.g. 6.8), and a different ionic content. This allows the proteins in a loaded sample to be concentrated into a tight band during the first few minutes of electrophoresis before entering the resolving portion of a gel. A stacking gel is not necessary when using a gradient gel, as the gradient itself performs this function.
The individual layers are prepared in situ in glass tubes by polymerizing a gel monomer and a cross-linking agent with the aid of an appropriate catalyst. The first gel to be poured into the tubular shape electrophoresis cell is the small pore separation gel. After gelation takes place (usually 30 minutes), a large pore gel (the spacer gel) is cast on top of the separation gel. Then a large pore monomer solution containing approximately 3 μL of serum is polymerized above the spacer gel, so that the finished product is composed of three different gel layers. When electrophoresis begins, all protein ions migrate unimpeded through the large pore gels and stack up along the end of the separation gel in a very thin zone. This process serves 121to concentrate protein components at the border (or starting) zone, so that the preconcentration of specimens with low-protein content (e.g. CSF) may not be necessary. Separation of the individual protein ions then occurs in the bottom separation gel, not only on the basis of their charges but also on the basis of their molecular sizes. The average pore size in a typical 7.5% PAGE separation gel is approximately 5 nm (50 Å). Protein whose molecular radius or length (or both) exceeds critical limits will be more or less impeded in their migration. Since both net charge and molecular size affect migration rate, serum proteins are fractionated into many zones. Acrylamide gel is thermostable, transparent, strong, and relatively chemically inert. It can be made in a wide range of pore sizes to optimize particular separations. Furthermore, these gels are uncharged, thus eliminating electroendosmosis. In one such procedure, simplifications of the original technique have been introduced. In one such procedure, the use of spacer and sample gels has been eliminated, a continuous buffer system replaces the discontinuous buffer system, and undiluted serum is delivered directly onto the top surface of the separation gel. Polyacrylamide gel is also used in isoelectric focusing.
Several forms of PAGE exist and can provide different types of information about the protein(s). Nondenaturing PAGE, also called native PAGE, separates proteins according to their mass-to-charge ratio. Denaturing and reducing SDS PAGE, the most widely used electrophoresis technique, separates proteins primarily by mass. Two-dimensional PAGE separates proteins by isoelectric point in the first dimension and by mass in the second direction.
Sodium dodecyl sulfate PAGE separates proteins primarily by mass because the ionic detergent SDS denatures and binds to proteins to make them evenly negatively charged. Thus, when a current is applied, all SDS bound proteins in a sample will migrate through the gel toward the positively charged electrode. Proteins with less mass travel more quickly through the gel than those with greater mass because of the sieving effect of the gel matrix.
Once separated by electrophoresis, proteins can be detected in a gel with various stains, transferred onto a membrane for detection by Western blotting and/or excised and extracted for analysis by mass spectrometry. Protein gel electrophoresis is, therefore, a common step in many kinds of proteomics analysis.
 
Native Polyacrylamide Gel Electrophoresis
In native PAGE, proteins are separated according to the net charge, size and shape of their native structure. Electrophoretic migration occurs, because most proteins carry a net negative charge in alkaline running buffers. The higher the negative charge density (more charges per molecule mass), the faster a protein will migrate. At the same time, the frictional force of the gel matrix creates a sieving effect retarding the movement of proteins according to their size and three-dimensional shape. Small proteins face only a small frictional force while large proteins face a larger frictional force. Thus, native PAGE separates proteins based upon both their charge and mass.
Because no denaturants are used in native PAGE, subunit interactions within a multimeric protein are generally retained and information can be 122gained about the quaternary structure. In addition, some proteins retain their enzymatic activity following separation by native PAGE. Thus, it may be used for preparation of purified, active proteins. Following electrophoresis, proteins can be recovered from a native gel by passive diffusion or electroelution. In order to maintain the integrity of proteins during electrophoresis, it is important to keep the apparatus cool and minimize the effects of denaturation and proteolysis. Extremes of pH should generally be avoided in native PAGE, as they may lead to irreversible damage to protein of interest, such as denaturation or aggregation.
 
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
In SDS PAGE, the gel is cast in buffer contain SDS and protein samples are heated with SDS before electrophoresis so that the charge density of all proteins is made roughly equal. Heating in SDS, an anionic detergent denatures proteins in the sample and binds tightly to the uncoiled molecule. Usually, a reducing agent such as dithiothreitol (DTT) is also added to cleave protein disulfide bonds and ensure that no quaternary or tertiary protein structure remains. Consequently, when these samples are electrophoresed, proteins separate according to mass alone, with very little effect from compositional differences.
When a set of proteins of known molecular weight are run alongside samples in the same gel, they provide a reference by which the mass of sample proteins can be determined. These sets of reference proteins are called molecular weight markers (MW markers) or standards, and they are available commercially in several forms. SDS PAGE is also used for routine separation and analysis of proteins, because of its speed, simplicity, and resolving capability.
 
Starch Gel Electrophoresis
Starch gel electrophoresis like PAGE separates macromolecular ions on the basis of both surface charge and molecular size. Partially hydrolyzed starch is used, since native starch does not gel. Starch gel may be used in a horizontal processor with migration taking place in the vertical direction. Preparation of gels is relatively difficult and requires considerable skill. The starch concentration is 10–16 g/dL, and the pH of the buffer varies according to the specific application.
 
Isoelectric Focusing Electrophoresis
Isoelectric focusing electrophoresis (IEFE) is a further advance in the electrophoretic separation of proteins into discrete bands. The medium provides a stable pH gradient by distributing carrier ampholyte along its axis. The compounds to be separated (e.g. proteins) migrate to the zone in the medium where the pH is equal to the isoelectric point of each compound. In that zone, the charge on the protein becomes zero and migration ceases. Figure 14 illustrates the process and shows the electrophoretic conditions 123before and after current is applied.
Fig. 14: Isoelectric focusing electrophoresis. Column 1: A stable pH gradient is established in the gel after application of an electric field. Column 2: Protein solution is added and electric field is reapplied. Column 3: After staining, proteins are shown to be distributed along pH gradient according to their isoelectric point values
With IEFE, separated protein zones are very sharp because the isoelectric point of a protein is confined to a narrow pH range and because diffusion of the protein is counteracted by acquisition of charge, as it moves away from its isoelectric point position electrical forces then drive it back to its isoelectric point position. Proteins that differ in isoelectric point value by only 0.02 pH unit have been separated by IEFE.
The pH gradient is created by the use of amphoteric polyaminocarboxylic acids (carrier ampholyte), a group of compound with molecular weights from 300 to 1,000. The carrier ampholyte are used in mixtures containing 50–100 different compounds. Under electrophoretic conditions, the many different carrier ampholytes generate a pH gradient, as each of them reaches its individual isoelectric point. The anode is surrounded by a dilute acid solution and the cathode by a dilute alkaline solution. Since carrier ampholyte are generally used in relatively high concentrations, a high voltage (up to 2,000 V) is necessary. As a result, the electrophoretic matrix must be cooled. Depending on analytical conditions, the power required may be in the vicinity of 250 volts and use of a constant power supply is advisable. If IEFE is carried out with constant voltage, frequent adjustments of the voltage are necessary because of a drop in current as electrophoresis proceeds. The drop in current is due to lower conductivity of carrier ampholyte at their isoelectric points and to the creation of zones or pure water as electrophoresis progresses. With a constant power supply, manual resetting of voltage is avoided or minimized. Constant current power supplies are not customarily used for IEFE.
Several support media have been used for IEFE, including polyacrylamide, agarose, and cellulose acetate. PAGE IEFE is widely used in analytical work. The polyacrylamide gel must be optically clear and have a large enough pore 124size so that, ideally, protein migration is unaffected by molecular sieving. In practice, impediment of migration of some proteins, characteristically of IgM, is unavoidable. Agarose and cellulose acetate products that are free of electroendosmotic effects have been adapted for AGE IEFE and CAE IEFE. These adaptations have the advantage of simple operating conditions, and their media have a large enough pore size to make the exclusion of proteins on the basis of molecular weight and size unlikely.
 
Precast Gels
Traditionally, researchers “poured” their own gels using standard recipes that are widely available in protein methods books. Most laboratories now depend on the convenience and consistency afforded by commercially available, ready to use, precast gels. Precast gels are available in a variety of percentages, including difficult to pour gradient gels that provide excellent resolution and separate proteins over the widest possible range of molecular weights. Technological innovations in buffer and gel polymerization methods enable manufacturers to produce gels with greater uniformity and longer shelf life than with traditional equipment and methods. In addition, precast polyacrylamide gels obviate the need to work with the acrylamide monomer—a known neurotoxin and suspected carcinogen.
 
One-dimensional Gel Electrophoresis
Comparative analysis of multiple samples is accomplished using 1D electrophoresis. Gel sizes range from 2 × 3 cm (tiny) to 15 × 18 cm (large format). The most popular size (8 × 10 cm) is usually referred to as a “mini gel”. Small gels require less time and reagents than their larger counterparts and are suited for rapid screening. However, larger gels provide better resolution and are needed for separating similar proteins or a large number of proteins.
Samples are added to sample wells at the top of the gel. When the electrical current is applied, the proteins move down through the gel matrix, creating what are called “lanes” of protein “bands”. Samples that are loaded in adjacent wells and electrophoresed together are easily compared to each other after staining or other detection step. The intensity of staining and “thickness” of protein bands are indicative of their relative abundance. The position (height) of bands within their respective lanes indicates their relative sizes (and/or other factors affecting their rate of migration through the gel).
 
Two-dimensional Electrophoresis
Multiple components of a single sample can be resolved most completely by 2D PAGE. The first dimension separates proteins according to their native isoelectric point using a form of electrophoresis called IEFE. The second dimension separates by mass using ordinary SDS PAGE. 2D PAGE provides the highest resolution for protein analysis and is an important technique in proteomic research (Fig. 15), where resolution of thousands of proteins on a single gel is sometimes necessary.125
Fig. 15: Overview of 2D gel electrophoresis. In the first dimension (left), one or more samples are resolved by isoelectric focusing (IEFE) in separate tube or strip gels. IEFE is usually performed using precast immobilized pH gradient (IPG) strips on a specialized horizontal electrophoresis platform. For the second dimension (right), a gel containing the isoelectric point resolved sample is laid across to top of a slab gel so that the sample can then be further resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE)
To perform IEFE, a pH gradient is established in a tube or strip gel using a specially formulated buffer system or ampholyte mixture. Readymade IEFE strip gels (called immobilized pH gradient strips or IPG strips) and required instruments are available from certain manufacturers. During IEFE, proteins migrate within the strip to become focused at the pH points at which their net charges are zero. These are their respective isoelectric points.
The IEFE strip is then laid sideways across the top of ordinary 1D gel, allowing the proteins to be separated in the second dimension according to size.
 
Immunofixation Electrophoresis
An agarose gel electrophoresis first separates the proteins in a serum sample. Antiserum against the protein of interest is spread directly on the gel. The protein of interest precipitates in the gel matrix. After a wash step to remove other proteins, the precipitated protein is stained. This method is qualitative and is used to identify proteins found in multiple myeloma.
Figure 16 shows the IFE gel from a serum sample analyzed on IFE agarose gel. After electrophoresis, the precipitated proteins are stained with acid violet. The SP lane represents a routine (SPE) of this specimen. On the next three protein separations, antiserum against IgG, IgA and IgM were applied to the G, A, M lanes, respectively. Antiserum to κ light chain was added to the next protein separation and antiserum to λ light chain to the last protein separation.126
Fig. 16: A serum sample analyzed on immunofixation electrophoresis agarose gel
Automated systems for protein electrophoresis are available for large volumes of samples for electrophoresis. An automated system is capable of separating 10–100 samples simultaneously. There are several different automated systems, and the number of process steps, which are automated varies. Automated steps may include reagent addition, sample application, electrophoresis separation, staining and detection.
 
Capillary Electrophoresis
 
Instrumentation
The instrumentation needed to perform capillary electrophoresis is relatively simple. A basic schematic of a capillary electrophoresis system is shown in Figure 17.
The system's main components are a sample vial, source and destination vials, a capillary, electrodes, a high voltage power supply, a detector, and a data output and handling device. The source vial, destination vial, and capillary are filled with an electrolyte, such as an aqueous buffer solution.
Fig. 17: An schematic diagram of capillary electrophoresis system
127
To introduce the sample, the capillary inlet is placed into a vial containing the sample and then returned to the source vial (sample is introduced into the capillary via capillary action, pressure, or siphoning). The migration of the analytes is then initiated by an electric field that is applied between the source and destination vials and is supplied to the electrodes by the high voltage power supply. It is important to note that all ions, positive or negative, are pulled through the capillary in the same direction by electroosmotic flow (EOF), as will be explained. The analytes separate, as they migrate due to their electrophoretic mobility, as will be explained, and are detected near the outlet end of the capillary. The output of the detector is sent to a data output and handling device, such as an integrator or computer. The data are then displayed on an electropherogram, which reports detector response as a function of time. Separated chemical compounds appear as peaks with different migration times in an electropherogram.
 
Modes of Separation
The separation of compounds by capillary electrophoresis is dependent on the differential migration of analytes in an applied electric field. The electrophoretic migration velocity (Up) of an analyte toward the electrode of opposite charge is:
where, μp is the electrophoretic mobility and E is the electric field strength. The electrophoretic mobility is proportional to the ionic charge of a sample and inversely proportional to any frictional forces present in the buffer. When two species in a sample have different charges or experience different frictional forces, they will separate from each other, as they migrate through a buffer solution. The frictional forces experienced by an analyte ion depend on the viscosity (η) of the medium and the size and shape of the ion. Accordingly, the electrophoretic mobility of an analyte at a given pH is given by:
where, Z the net is charge of the analyte and r is the Stokes radius of the analyte. The Stokes radius is given by:
where, kB is the Boltzmann constant, T is the temperature, and D is the diffusion coefficient. These equations indicate that the electrophoretic mobility of the analyte is directly proportional to the charge of the analyte and inversely proportional to its radius. The electrophoretic mobility can be determined experimentally from the migration time and the field strength:
where, L is the distance from the inlet to the detection point, tr is the time required for the analyte to reach the detection point (migration time), V is the applied voltage (field strength), and Lt is the total length of the capillary.128
Fig. 18: Diagram of the separation of charged and neutral analytes (A) according to their respective electrophoretic and electroosmotic flow mobilities
Since only charged ions are affected by the electric field, neutral analytes are poorly separated by capillary electrophoresis.
The velocity of migration of an analyte in capillary electrophoresis will also depend upon the rate of EOF of the buffer solution. In a typical system, the EOF is directed toward the negatively charged cathode so that the buffer flows through the capillary from the source vial to the destination vial. Separated by differing electrophoretic mobilities, analytes migrate toward the electrode of opposite charge. As a result, negatively charged analytes are attracted to the positively charged anode, counter to the EOF, while positively charged analytes are attracted to the cathode in agreement with the EOF as depicted in Figure 18.
The velocity of the EOF Uo can be written as:
where, μo is the electroosmotic mobility that is defined as:
where, ζ is the zeta potential of the capillary wall, and ε is the relative permittivity of the buffer solution. Experimentally, the electroosmotic mobility can be determined by measuring the retention time of a neutral analyte. The velocity U of an analyte in an electric field can then be defined as:
Since the EOF of the buffer solution is generally greater than that of the electrophoretic flow of the analytes, all analytes are carried along with the buffer solution toward the cathode. Even small, triply charged anions can be redirected to the cathode by the relatively powerful EOF of the buffer solution. Negatively charged analytes are retained longer in the capillary due to their conflicting electrophoretic mobilities. The order of migration seen by the detector is shown in Figure 18 small multiple charged cations migrate quickly, and small multiple charged anions are retained strongly.
Electroosmotic flow is observed when an electric field is applied to a solution in a capillary that has fixed charges on its interior wall. Charge is accumulated on the inner surface of a capillary when a buffer solution is 129placed inside the capillary.
Fig. 19: Depiction of the interior of a fused silica gel capillary in the presence of a buffer solution
In a fused silica capillary, silanol (SiOH) groups attached to the interior wall of the capillary are ionized to negatively charged silanoate (SiO) groups at pH values greater than three. The ionization of the capillary wall can be enhanced by first running a basic solution, such as NaOH or KOH through the capillary prior to introducing the buffer solution. Attracted to the negatively charged silanoate groups, the positively charged cations of the buffer solution will form two inner layers of cations (called the diffuse double layer or the electrical double layer) on the capillary wall as shown in Figure 19.
The first layer is referred to as the fixed layer, because it is held tightly to the silanoate groups. The outer layer, called the mobile layer, is farther from the silanoate groups. The mobile cation layer is pulled in the direction of the negatively charged cathode when an electric field is applied. Since these cations are solvated, the bulk buffer solution migrates with the mobile layer, causing the EOF of the buffer solution. Other capillaries, including Teflon capillaries also exhibit EOF. The EOF of these capillaries is probably the result of adsorption of the electrically charged ions of the buffer onto the capillary walls. The rate of EOF is dependent on the field strength and the charge density of the capillary wall. The wall's charge density is proportional to the pH of the buffer solution. The EOF will increase with pH until all of the available silanols lining the wall of the capillary are fully ionized.
 
Efficiency and Resolution
The number of theoretical plates, or separation efficiency, in capillary electrophoresis is given by:
where, N is the number of theoretical plates, μ is the apparent mobility in the separation medium, and Dm is the diffusion coefficient of the analyte. According to this equation, the efficiency of separation is only limited by diffusion and is proportional to the strength of the electric field. The efficiency of capillary electrophoresis separations is typically much higher than the efficiency of other separation techniques like high performance liquid 130chromatography (HPLC).
Fig. 20: Flow profiles of laminar and electroosmotic flow
Unlike HPLC in capillary electrophoresis, there is no mass transfer between phases. In addition, the flow profile in EOF driven systems is flat, rather than the rounded laminar flow profile characteristic of the pressure driven flow in chromatography columns as shown in Figure 20. As a result, EOF does not significantly contribute to band broadening as in pressure driven chromatography. Capillary electrophoresis separations can have several hundred thousand theoretical plates.
The resolution (Rs) of capillary electrophoresis separations can be written as:
According to this equation, maximum resolution is reached when the electrophoretic and electroosmotic mobilities are similar in magnitude and opposite in sign. In addition, it can be seen that high resolution requires lower velocity and, correspondingly, increased analysis time.
A family of related separation techniques that use narrow bore fused silica capillaries to separate a complex array of large and small molecules. High voltages are used to separate molecules based on differences in charge, size and hydrophobicity. Injection into the capillary is accomplished by immersing the end of the capillary into a sample vial and applying pressure, vacuum, or voltage. Depending on the types of capillary and buffers, capillary electrophoresis can be segmented into several separation techniques. Examples of these include the following:
 
Capillary Zone Electrophoresis
Also known as free solution capillary electrophoresis (FSCE), it is the simplest form of capillary electrophoresis (Fig. 21). The separation mechanism is based on differences in the charge-to-mass ratio of the analytes. Fundamental to capillary zone electrophoresis (CZE) are homogeneity of the buffer solution and constant field strength throughout the length of the capillary. The separation relies principally on the pH controlled dissociation of acidic groups on the solute, or the protonation of basic functions on the solute.
Capillary zone electrophoresis analytes move in the EOF but separate into bands because of differences in their electrophoretic mobilities, (μ).131
Fig. 21: Capillary zone electrophoresis
Differences in μ make each analytes overall migration velocity slightly different, and difference in migration velocity, i.e. separation. Electrophoretic mobilities are roughly a function of analyte charge and frictional and size differences. In Figure 22 three peaks are traveling down the capillary from the beginning of the capillary on the left to the detector and exit reservoir on the right (again, this is a simple schematic). In this system, an absorption detector would work if the analytes have good molar absorption at wavelengths the detector has available (usually in the ultraviolet). If the analytes are poor absorbers then a strong ultraviolet absorber can be added to the run buffer and the decrease in absorption—when the analytes pass the detector—can be used to detect the analytes. This last is called indirect absorption. Figure 22A, where the three analytes are still on the capillary; the electropherogram results, which plots time versus signal, shows a flat line against Figure 22B. Only when the analytes arrive at the detector will peaks appear in the electropherogram. Note: In CZE, there is buffer between analyte bands. “All” that's required in the CZE method is a well-chosen buffer in the initial buffer reservoir. Separation occurs because of relatively simple interaction of the analytes with the pH of the buffer. This technique is also called FSCE for that reason. The capillary is often prewashed with the buffer; the sample—dissolved in the same buffer—is injected; EOF is established; and you're off. This should be contrasted with the methods discussed below. In the Figure 22B, two of the peaks have already passed the detector and the third is about to.
Capillary gel electrophoresis (CGE) is the adaptation of traditional gel electrophoresis into the capillary using polymers in solution as a molecular sieve. This allows solutes having similar charge-to-mass ratios to be resolved by size (Fig. 23).
Capillary isoelectric focusing (CIEF) allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode (Fig. 24). A solute will migrate to a point 132where its net charge is zero.
Figs 22A and B: A. Electrophoresis relationship between time and detector signal; B. Emergence of multiple peaks.
Fig. 23: Capillary gel electrophoresis
At this isoelectric point (the solute's pI), migration stops and the sample is focused into a tight zone. In CIEF, once a solute has focused at its pI, the zone is mobilized past the detector by either pressure or chemical means.
The buffer in CIEF is arranged in a pH gradient; it is usually a commercial mixture of so called carrier ampholytes, many different zwitterions with a range of isoelectric points. These molecules are also small, so their electrophoretic mobilities are high, and they can move quickly. The more individual (that is different) ampholytes there are in the buffer, the smoother the pH gradient will be; and similarly, the smoother the pH gradient, the better the separation 133between closely eluting peaks.
Fig. 24: Capillary isoelectric focusing
After filling the capillary with a mixture of the ampholytes and analyte molecules, a strong base is placed in one “buffer” reservoir and a strong acid in the other. When the system's electrical potential is applied, just as if we were trying to establish EOF, all molecules start to migrate to their isoelectric point, but the carrier ampholytes move quickest because their mobility is very high compared to the analytes’ (let's say the analytes are protein), and this means that the pH gradient is established after a short time; the time length is a function of the carrier ampholytes characteristic and the voltage applied. This also applies to the focus step next: higher voltages decreases analysis time. But after the carrier ampholytes have reached their isoelectric points and the pH gradient has been established, the (slower moving) analytes are still moving to get to their isoelectric points. Each analyte molecule also has a different isoelectric point (combination of multiple Kas or Kbs for each of these macromolecules) so analyte bands focus at different capillary locations in the pH gradient during this, the so called “focusing step.” In theory, the process is complete when “everyone” has reached their respective isoelectric points. And also in theory, this can be followed by watching the current in the system drop to zero. Remember that current is a measure of charge flow in the circuit and when all ions are at their isoelectric point—and stop moving—the charge flow in the electrophoretic circuit stops, so the current should fall to zero. In reality, analysts seldom wait for this; based upon previous experience, they may wait until the initial current has dropped to, say, 20% of the initial value and then the run is stopped. Running at the highest voltage possible yields the fastest run as long as the heat generated can be dissipated. Modern instruments can also be programmed to run at a constant power, so as the current drops as ions stop moving, the voltage ramps up automatically to compensate, but this too is also usually run until some predetermined low current flow is achieved, and then the run stopped. After analyte focusing, the EOF is begun by changing the ionic strength in one of the buffer reservoirs; focused analytes and all 134ampholytes move to the detector. Note, in the Figure 24 that the peaks have reached their isoelectric points and are still far away from the detector, and therefore the electropherogram shows no eluted peaks, yet. They will then move at that same spacing/separation toward the detector. In the adjacent Figure 24, the first peak is just about to finish eluting and is reflected in the chromatogram. Finally, peak identification can be accomplished in CIEF by using chemical markers of known isoelectric points. Analytes will then be spaced before or after these markers in the electropherogram. Marker compounds can be added to the sample in amounts that help them to be identified in the subsequent electropherogram.
 
Capillary Isotachophoresis
Capillary isotachophoresis (CITP) sample injections are preceded by high-mobility ions (H) and followed by low-mobility ions (L) chosen so that analyte conductivities/mobilities lie between μ of H and L. This means that the H electrolyte must have a higher mobility than any of the analyte ions, and L must have mobility lower than any of the analyte ions. This is one step more complicated than CZE in that more careful solutions must be prepared than merely the run buffer of CZE. The capillary is first filled with a solution containing the leading electrolyte (H). Leading that is high mobility, ions can be small, completely dissociated ions, such as Cl for anionic separations or K+ for cationic separations. Then analyte ions (the sample) are injected. Next, a solution containing the trailing, that is low mobility, electrolyte (L) is introduced into the capillary's entrance reservoir and the capillary inserted. Trailing ions can be a weak acid in the case of anions separations or a weak base for cationic separation. In this way, the analytes are sandwiched between H and L. When EOF is established, the analytes achieve a separation order based upon their mobilities, which also corresponds to their relative conductivities: highest mobile ions (H) are most conductive and lowest (L) ones are least conductive, analytes inbetween. One of the most interesting features about this method of capillary electrophoresis is that the analytes order themselves immediately next to each other, i.e. there is no buffer between analyte bands. This makes the CITP electropherogram very interesting (well, interesting for “traditional” chromatographers). So after the quick ordering of the “ion sandwich” EOF moves all ions past the detector and off the column. CITP analytes also move in the EOF—after their quick separation all at the same speed (isotach = same speed). Unlike CZE and CIEF, there is no buffer between analyte bands in CITP. Since the current (I) is consistently maintained across the entire capillary, the resistance of each analyte establishes a different potential, V, in each analyte band—the separating force. To satisfy V = IR, bands quickly form, ordered from bands of lowest to highest R. Band broadening is minimized in CITP because there is no buffer between analyte bands, and if analytes near a band's edge diffuse into the adjacent band (longitudinal diffusion) they experience a different voltage (V = IR again) and move back into their own band. And finally the “identity” of each analyte is a function of its conductivity (the conductivity detector's signal intensity, y axis) and the amount of the analyte is a function 135of the width of the analyte band (x axis).
Fig. 25: Capillary Isotachophoresis
Fig. 26: Nonaqueous capillary electrophoresis
Note the stair step feature of the CITP data: the fastest mobility and highest conductivity ions elute first (H) and each analyte after that is more resistant (less conductive) and less mobile until the least mobile solution (L) elutes.
This is an easy way to recognize CITP data from a conductivity detector, if the detector were not a conductivity detector then the electropherogram would look more “normal” (with baseline return of the detector signal after a peak) but there is a good chance that many of the analytes would have no measurable ultraviolet absorption, and so would be missing from the electropherogram. That is why CITPs power is best released by a conductivity detector for many applications (Fig. 25).
Nonaqueous capillary electrophoresis (NACE) involves the separation of analytes in a medium composed of organic solvents. The viscosity and dielectric constants of organic solvents affect both sample ion mobility and the level of EOF (Fig. 26). The use of nonaqueous medium allows additional selectivity options in methods development and is also valuable for the separation of water insoluble compounds.136
 
Quality Control
The laboratory is responsible for providing accurate responses and must ensure as much as possible that they are interpreted and applied in the best interest of the patient. The quality of results begins with monitoring a well-functioning instrument. Using qualitative and/or quantitative agarose electrophoresis or using capillary electrophoresis, well-calibrated methods as well as regular use of an internal quality control material in each electrophoresis procedure will ensure the quality of the work. The internal quality control material is a pooled sera or commercially available quality control sera.
The internal quality control material should give percentages of albumin, α1, α2, β, and γ globulins, and the γ component being of normal distribution to be considered as a normal internal quality control, and not containing any monoclonal protein component. On the other hand, a pathological internal quality control of quantitative material containing a monoclonal protein at decision level for treating myeloma patients would be beneficial to all laboratories as an SPE control, whether the analysis is by agarose electrophoresis or a capillary electrophoresis. The same applies for control material for urinary or CSF protein electrophoresis.

Interpretive Guide of Individual Specific Proteins Electrophoresis of Normal and Abnormal PatternsCHAPTER 5

“Specialist actions in selection and interpretation of the tests are part of the service which the laboratory provides”.
The following serum protein electrophoresis reference values are representative. Some variations among laboratories and specific methods are to be expected. The values were obtained by standard electrophoresis on agarose gels (Fig. 1).
  • Total protein: 6.4–8.3 g/dL (about 0.5 g/dL lower in nonambulatory patients)
  • Albumin: 3.5–5.0 g/dL
  • α-1 globulin: 0.1–0.3 g/dL
  • α-2 globulin: 0.6–1.0 g/dL
  • β-globulin: 0.7–1.2 g/dL
  • γ-globulin: 0.7–1.6 g/dL.
Albumin levels are increased in dehydration and decreased in malnutrition, pregnancy, liver disease, inflammatory diseases, and such protein losing states as malabsorption syndrome and certain kidney disorders.
α 1 globulins are increased in inflammatory diseases and decreased or absent in juvenile pulmonary emphysema, a hereditary disease. α-2 globulins are increased in acute and chronic inflammation and nephrotic syndrome; decreased values may indicate hemolysis.
β globulin levels are increased in conditions of high cholesterol (hypercholesterolemia), in multiple myeloma, and in iron deficiency anemia, and decreased in disorders associated with complement depletion.
γ globulin levels are increased in chronic inflammatory disease and such autoimmune conditions as rheumatoid arthritis and systemic lupus erythematosus, cirrhosis, in acute and chronic infection and in multiple myeloma. The γ globulins are decreased in a variety of genetic immune 138disorders, in secondary immune deficiency related to steroid use, leukemia, or severe infection.
Fig. 1: Serum protein
Detection of a discrete (monoclonal) band in the γ region of the electropherogram indicates the presence of a paraprotein. Type IgG or IgA paraproteins associated with multiple myeloma may be found by serum protein electrophoresis testing; however, the tumor may produce only Ig light chains that are removed from the blood by the kidneys. The Ig light chain (Bence Jones protein) can be detected by urine protein electrophoresis.
 
VISUAL INSPECTION
 
Serum Protein
 
Prealbumin
Prealbumin is a thin and quick indicator of inflammatory reaction:
  • Treatment with corticosteroids, use of anabolic steroids, Hodgkin's disease, alcoholism, and acromegaly
  • Hepatopathy, nephropathies, hereditary amyloidosis, hyperestrogenism, thyrotoxicosis, and intravenous administering of liquids (Fig. 2).
 
Albumin
A general indicator of the polyclone state of the organism, its reliability is compromised by: 139
Fig. 2: Prealbumin band (arrow)
Fig. 3: Hyperalbuminemia (arrow)
  • Long half-life (19 days)
  • Decreased synthesis during most inflammatory processes
Currently: prognostic value, in particular in chronic patients.
  • Hemoconcentration
 
Hyperalbuminemia
In healthy individuals, the presence of hyperalbuminemia does not necessarily have any pathological meaning. It can be seen usually in hospitalized patients, due to hemoconcentration (by dehydration) or albumin administration (Fig. 3).
 
Bisalbuminemia
Double band is seen on the electrophertogram as an albumin fraction split in two, and it can be of permanent or transient nature. It is a result of:
  • Hereditary mutation: the double band is then a permanent sign of a genetic variant, generally without any observed pathological effect
  • Acquired transient bisalbuminemia: it occurs due to pancreatitis or a drug treatment, such as high doses of β lactam in a patient with renal insufficiency, through binding of the antibiotic to albumin (Figs 4 and 5).
 
Hypoalbuminemia
Since albumin is exclusively of hepatic origin, any decrease in the percentage of albumin is the result of one of the following mechanisms:
  • Severe chronic malnutrition140
    Fig. 4: Bisalbuminemia condition (arrow)
    Fig. 5: Bisalbuminemia
  • A decrease in synthesis—lymph proliferative disorder, hepatocellular insufficiency (cirrhosis, hepatitis), inflammation
  • Increased losses—urinary (nephrotic syndrome), digestive (exudative gastroenteropathy) or cutaneous (widespread burns)
  • Hypercatabolism—acquired endocrine disorders (thyrotoxicosis, Cushing's disease), severe inflammatory syndromes (Fig. 6).
 
Congenital Analbuminemia
For rarely occurring cases of analbuminemia, the electrophoretic pattern is unusual (very low albumin band). The four globulin fractions increase in order to keep the osmotic pressure as high as possible. However, clinical symptoms are usually limited to discrete edema (Figs 7 to 9).
 
Changes in the α-Globulin Zone
 
Decrease
  • Brought about by hepatocellular insufficiency, malnutrition or protein loss, generally with concomitant decrease of albumin, α2 and β globulins141
    Fig. 6: Normal albumin band (thin arrow) and hypoalbuminemia (thick arrow)
    Fig. 7: Analbuminemic condition (arrow)
    Fig. 8: Analbuminemia
    Fig. 9: Nephrotic syndrome
    142
  • Caused by congenital deficiency of α1 antitrypsin, the predominant protein in α1 zone.
Such deficiencies are due to specific alleles of the α1 antitrypsin gene. Pi* S, Pi* Z or Pi* null that may be partially compensated when associated in a heterozygous state with an allele expressed at a normal level. This abnormality is sometimes associated with liver and lung diseases (emphysema).
 
α-1 Antitrypsin
Clinical significance: Congenital deficiency is associated with the development of hepatomegaly, young cirrhosis, obstructive lung disease, emphysema; in subjects of Northern European origins increased occurrence of hepatoma is verified. Evaluation of the risk in familial studies (phenotypes MZ, SS, SZ, ZZ)
  • Pregnancy, treatment with estrogens or androgens, acute hepatitis, acute liver disease (including alcoholism), neoplasia
  • Congenital defect, nephrotic syndrome, terminal stage hepatic or pancreatic disease and malnutrition (Figs 10 to 13).
 
α-2 Macroglobulin
Clinical significance: Key role in rapid control and early removal of protease activated in circle (cascade of coagulation, complement, collagenase, lysosomal cathepsins, etc.) involved in the transport of cytokines, hormones and metals (zinc in particular), indicator of the permeability of membrane in the serum and other bodily fluids.
Fig. 10: Moderate increment of α1 antitrypsin (arrow)
Fig. 11: Heterozygosis α1 antitrypsin
143
Fig. 12: α1 antitrypsin (thin arrow) and total α1 antitrypsin deficient (thick arrow)
Fig. 13: α1 antitrypsin deficient
Double α2 globulin band can occur in the following cases:
  • In vitro hemolysis: hemoglobin if present in the sample is migrating in the α2 zone (complexed to haptoglobin)
  • The presence of specific phenotypes of haptoglobin (Hp 1-1) shows different electrophoretic mobility than Hp 1-2 or 2-2
  • More rarely the presence of β lipoprotein [low density lipoproteins (LDL)] of α-2 abnormal electrophoretic mobility, or
  • Presence of a monoclonal free light chain migrating in this area (Fig. 14).
 
Increase
This increase is mainly seen in two types of syndromes and is related to the variable level of the two main proteins migrating into the α2 globulin zone:
  • The inflammatory syndrome, by an increase of haptoglobin (the α2 fraction is then greater than 15%), associated with hyper α1 globulinemia
  • The nephrotic syndrome, by an often substantial increase of α2 macroglobulin associated with hypoalbuminemia (due to urinary loss), hyper β globulinemia (in particular lipoid nephrosis) and with proteinuria exceeding 3 g/L (Figs 15 and 16).144
Fig. 14: Double α2 globulin band (arrows)
Fig. 15: Increased (thin arrow) and normal (thick arrow) α2 macroglobulin
 
Decrease
  • Brought about by hepatocellular insufficiency, malnutrition or protein loss (Fig. 17)
  • By intravascular hemolysis: the fall in haptoglobin will be even more visible in the protein electrophoresis if an associated inflammatory syndrome exists (discrepancy between the increase in the α1 zone and the decrease in the α2 zone).
 
Changes in the β-Globulin Zone
 
Increase
The causes may vary according to the extent of the increase:
  • Hyper β globulinemia by hypertransferrinemia in anemia or by increased β lipoprotein (Fig. 18)
  • Nonmonoclonal causes (usually limited increase): hyper β2 globulinemia by increased C3 inflammatory or secondary hyper β2 globulinemia due to intra- or extrahepatic biliary obstruction
  • β-zone elevated as a whole and associated with a β-γ bridge, thus revealing the polyclonal hyperimmunoglobulin A observed in alcoholic cirrhosis (Fig. 19).
  • Monoclonal proteins:
    • Monoclonal immunoglobulin G or A (the most frequent) (Fig. 20)145
      Figs 16A and B: A. Acute; B. Chronic inflammatory panel
      Fig. 17: Normal (thin arrow) and decreased (thick arrow) α-2 macroglobulin (thick arrow)
      Fig. 18: Increment in β globulin (arrow)
      146
      Fig. 19: β-γ bridge
      Fig. 20: Monoclonal in β region
    • Monoclonal immunoglobulin M (WaldenstrÖm's disease)
    • K or λ monoclonal free light chains, seen in light chain myeloma or amyloidosis.
 
Decrease
  • Induced by hepatocellular insufficiency, malnutrition or protein loss related to a decrease in transferrin migrating into the β1 zone (Fig. 21)
  • Induced by C3 consumption associated with a decrease in the β2 zone; the decrease of β2 can be due to ageing of the serum sample.
 
β Lipoprotein (Apo B)
Clinical significance: Electrophoresis of lipoproteins is a simple method, useful for studying dyslipidemia. Their differentiation based on the electrophoretic mobility is the basis of the Fredrickson classification. This classification allows us to quickly prepare a dietetic or therapeutic treatment.
The β-lipoproteins or LDL migrate normally in cathodic or anodic position of the β-1 globulins. When the polyclones component reaches the level of sensitivity of the coloring the presence can be observed (Fig. 22).147
Fig. 21: Decrease of β2 zone (arrow)
Fig. 22: No β-lipo band (thin arrow) and β-lipo is recognizable due to its form in curly brackets or (zigzagging) (thick arrow)
 
Transferrin
Clinical significance: Iron transport glycoprotein usually saturated at one-third of its bonding capacity. In conditions of sideropenia its increase precedes anemization by days or months. It is useful in differential evaluation of anemia and in evaluation of patients at risk of iron overload.
  • Sideropenia, acute hepatitis, pregnancy and treatment with estrogens, hypothyroidism
  • Neoplasia, hepatopathies, nephrotic syndrome, malnutrition, conditions of iron overload, congenital atransferrinemia, patients in dialysis, chronic kidney insufficiency (Figs 23 to 25).
 
Cryoglobulin
Cryoglobulinemic syndrome (SC), the result of a protracted antigenic stimulus, is a sign of immune dysregulation, the genesis of which seems to be multifactorial, because it is accompanied by much diversified pathologies. In recent years the close association between infection by hepatitis C virus, the presence of mixed cryoglobulins (CR), and development of SC has been widely demonstrated and the etiopathogenetic base of this syndrome 148has been clarified—a systemic leukocytoclastic vasculitis, mediated by cryoprecipitate immunocomplexes; immunocomplexes, which in EGFR-specific transcription factor precipitate, in the link of the support, during the phase of application of the biological sample.
Fig. 23: Transferrin: A. Normal; B. Decreased
Fig. 24: Increased (thin arrow) and normal (thick arrow) transferrin
Fig. 25: Hypertransferrinemia
This event determines the appearance of a homogeneous band collapsed near the point of application. The morphology of the band is recognizable by the clear and net margins, its shape is always very serrated on the anode-cathode axis, and the intensity of coloring is dark and brilliant (Fig. 26).149
Fig. 26: Cryoglobulinemic immunocomplexes (thin arrow) and without the presence of immunocomplexes (thick arrow)
Fig. 27: C4 located between transferrin (increased) and much reduced C3 (thin arrow) and absence of C4 and strong consumption of C3 (thick arrow)
 
Complement Fraction 4
Clinical significance: C4 is part of the classic C3 converted path and, therefore, its diagnostic significance follows that cited for C3.
C4 is a 206 KD β1 globulin comprised of three unequal chains bonded by disulfide bridges. Serine esterase removes a biologically active peptide (C4a) and the remainder of the molecule (C4b) acquires the capacity to adhere to cellular membranes or bacterial walls (Fig. 27).
 
Complement Fraction 3
Clinical significance: Decreased levels indicate activation of the complement, decreased synthesis, protein loss or consumption (i.e. in different autoimmune diseases). The total or partial deficiency of C3 is associated with recurring infections and increased risk of lower esophageal sphincter [lupus erythematosus (LES)]. Useful in monitoring the progression and/or clinical activity of some autoimmune diseases (i.e. acute glomerulonephritis)
  • APR (delayed), biliary obstruction, obstructive jaundice, diabetes mellitus, gout, connectival diseases (not including LES)150
    Fig. 28: Normal C3 band (thin arrow) and depressed C3 by consumption (thick arrow)
    Fig. 29: β-2 microglobulin (thin arrow) and No β microglobulin (thick arrow)
  • Autoimmune diseases, immunocomplex diseases, mixed cryoglobulinemia, advanced hepatopathia, chronic kidney insufficiency, familial hemolytic- uremic syndrome, thrombotic thrombocytopenic purpura, congenital deficiency (Fig. 28).
 
β2 Microglobulin
This protein has aroused much interest for two distinct reasons: (i) for its extraordinary homology of sequence with respect to the immunoglobulins and (ii) for its association with the antigens of human leukocyte antigen histocompatibility (Fig. 29).
 
Fibrinogen
In the screening investigation the use of correctly withdrawn plasma samples allows us to obtain, with respect to the serum, a greater amount of information of clinical interest, since fibrinogen represents a very sensitive index of inflammation. Upon a panoramic examination of the electrophoretic track, it is often precisely the intensity of the band of fibrinogen, which attracts attention to the existence of a phlogistic syndrome. For clinical purposes, the visual evaluation of the track provides advantages with respect to the isolated determination of the fibrinogen. Increase of this protein in the absence of 151electrophoretic signs of inflammation can be due to affections with increased capillary permeability.
Fig. 30: Fibrinogen band (thin arrow) and absence of fibrinogen
The confirmation, on the other hand, in a track with evident signs of phlogistic syndrome of a weak band of the fibrinogen must lead to suspect the existence of acute or subacute inflammations accompanied by consumption or increased fibrinolysis. Particular morphological aspects of the band of fibrinogen can sometimes have diagnostic significance. It is the case, for example, of acute pancreatitis in which the normal band can be substituted by a hazy area with indistinct limits (Fig. 30).
 
Changes in the Gammaglobulin Zone
Hypergammaglobulinemia
  • Polyclonal hypergammaglobulinemia (diffuse increase) mainly observed in viral or bacterial infections, AIDS, or autoimmune diseases
  • Monoclonal hypergammaglobulinemia: sharp, narrow and homogeneous electrophoretic band, or bands if present under different polymerization forms, as a result of the presence of a monoclonal component
  • Biclonal hyperglobulinemia
  • Oligoclonal hyperglobulinemia (several narrow and homogeneous bands)
  • In specific cases, hyperglobulinemia arises from an increase in some subclasses resulting in a particular oligoclonal pattern
These immunoglobulins correspond either to:
  • Autoantibodies seen in some autoimmune diseases—rheumatoid arthritis, SjÖgren's syndrome, lupus erythematosus, progressive systemic sclerosis, etc.
  • Antibodies directed against viral proteins—seropositive individuals with HIV, viral hepatitis, meningitis, cytomegalovirus infections
  • Autoimmune responses in transplanted patients on immunosuppressive therapy
  • Immune responses in normal individuals: 1–5% of normal individuals may show an oligoclonal pattern of no clinical value. The monoclonal type bands are often present in low concentration, and usually transient (Fig. 31).
 
Polyclonal Structures
The polyclonal structures have been shown in Figures 32 to 34.152
Fig. 31: Normal gammaglobulin (thin arrow) and hypergammablobulin (thick arrow)
Fig. 32: Polyclonal structure of the immunoglobulin at the upper limits of the reference (thin arrow), average limit of the reference area (thick arrow) and at the lower reference area (thick dashed arrow)
Fig. 33: Polyclonal structure of the immunoglobulin showing β-γ bridge
153
Fig. 34: Polyclonal hyperglobulinemia
Fig. 35: IgA K monoclonal in α2 region (arrow)
Fig. 36: Presence of mesangial cells IgA λ in α2 interarea-transferrin (arrow)
Fig. 37: Monoclonal band adjacent to C3 area (arrow)
154
Fig. 38: Monoclonal band in γ central area (arrow)
Fig. 39: Monoclonal bands in γ area with polyclonal base medium conserved (arrows)
Fig. 40: Monoclonal bands in γ area of which strongly retromigrated with depressed polyclonal base (arrows)
Fig. 41: Monoclonal band (arrow)
155
Fig. 42: Monoclonal component in subject with medium conservation of polyclonal immunoglobulin
Fig. 43: Monoclonal component
 
Monoclonal Immunoglobulin Structures
The monoclonal structures have been shown below in Figures 35 to 43.
Monoclonal bands in γ area of which strongly retromigrated with depressed polyclonal base.
The biclonal and oligoclonal profiles are given in Figures 44 and 45.
 
Hypogammaglobulinemia
  • Physiological hypogammaglobulinemia in babies
  • Isolated or total primary immunodeficiency (involving one or more immunoglobulin classes), in children and adults
  • Secondary hypogammaglobulinemia: associated with myeloma or due to corticosteroids and immunosuppressive treatments, chemotherapy or radiotherapy
  • Light chain myeloma hypogammaglobulinemia: the diagnosis will be confirmed by the detection of Bence Jones protein in the urine (Figs 46 and 47).
 
Immunoglobulin
 
Immunoglobulin G
Clinical significance: The IgG represents from 75% to 80% of the total Ig, fixes the complement through the classic path (IgG 1, IgG 2, IgG 3) and alternative 156path (IgG 4) and constitute the specific immune response.
Fig. 44: Biclonal profile
Fig. 45: Oligoclonal profile
Fig. 46: Hypogammaglobulin (thin arrow) and normal gammaglobulin (thick arrow)
It is are the only Ig that passes the placenta. Deficiency of IgG is associated with repeated serious infections.
  • Autoimmune diseases (LES, acute renal, systemic sclerosis, Sjogren's syndrome, etc.), chronic hepatopathy, recurrent or chronic infections, sarcoidosis, some, intrauterine contraceptive devices.
    Oligoclonal: Lymphoid and nonlymphoidal neoplasia, viral infections, autoimmune diseases.157
    Figs 47A to C: Hypogammaglobulinemia (arrows)
    Monoclonal: IgG myeloma monoclonal gammopathy of undetermined significance (MGUS), lymphomas.
    • Infancy, pregnancy (moderate), hypogammaglobulinemia, agammaglobulinemia, nephrotic syndrome, non IgG myelomas.
In the sample on the left of the α1 area is in a more anodic position with respect to the same protein in the sample on the right.
 
IgA
Clinical significance: The IgA fixes the complement by the alternative path; polyclonal increase of IgA can be present in chronic inflammatory panels of the respiratory tract and the gastrointestinal tract, including the liver. About one-fourth of the patients with IgA deficiency has anti-IgA antibodies 158and is at risk of serious anaphylactic reactions in case of blood/plasma transfusions.
  • Chronic hepatopathy, cirrhosis, chronic respiratory infections, neoplasia of the final section of the intestine, gastrointestinal apparatus diseases (Crohn's disease, ulcerative colitis, etc.), rheumatoid arthritis, ankylosing spondylitis, neuropathies, Wiskott-Aldrich syndrome, IgA myeloma, MGUS (monoclonal)
  • Infancy, selective deficiency of IgA (approximately 1/700), protide dispersion syndrome, macroglobulinemia, or non-IgA myeloma.
 
Immunoglobulin M
Clinical significance: The IgM fixes the complement through the classic path and constitutes the primary response to the infections. Viral IgM specific in newborns are due to congenital infections (they do not pass through the placenta filter).
  • Viral infections, parasitosis, chronic hepatopathy, primitive biliary cirrhosis, primary sclerosing cholangitis; monoclonal: WaldenstrÖm macroglobulinemia, malignant lymphoma, reticulosis, CRs, cryoagglutinins
  • Infancy, immunodeficiency conditions (Wiskott-Aldrich syndrome), non- IgM myeloma.
 
Immunofixation
The procedure of detection and typing of immunoglobulin or monoclonal antibodies in serum or urine has been shown in Figures 48 to 67.
 
The Bence Jones Protein
The diagnosis of Bence Jones Protein has been shown in Figures 68 to 76.
Fig. 48: Presence of 1 IgG λ mesangial cells
159
Fig. 49: Presence of 1 IgG κ mesangial cells
Fig. 50: Presence of 2 IgG κ mesangial cells. Good conservation of the polyclonals
Fig. 51: Presence of 2 IgG κ mesangial cells. Discrete conservation of the G polyclonals
160
Fig. 52: Presence of 2 IgG κ mesangial cells. Low conservation of the G polyclonals
Fig. 53: Presence of one very small IgG λ mesangial cells. The high concentration of the κ polyclonals can mislead in attribution of the type of light chain
161
Fig. 54: Presence of IgG class mesangial cells and λ type
Fig. 55: Presence of 2 IgG κ mesangial cells. Strong depletion of the polyclonals
162
Fig. 56: Presence of 2 IgG λ mesangial cells. Note the strong reduction of the polyclonal heterogeneity of the immunofixated IgA which simulates a compatible band with a “monoclonal” morphology
Fig. 57: Presence of 3 IgA λ mesangial cells
Fig. 58: Presence of 2 IgA λ mesangial cells in γ C3 position
163
Fig. 59: Presence of 2 mesangial cells in central γ position of IgM κ and λ respectively
Fig. 60: Presence of class IgM κ mesangial cells
Fig. 61: Presence of 2 IgM κ mesangial cells
164
Fig. 62: Presence of IgG λ mesangial cells and λ free light chains mesangial cells
Fig. 63: Presence of IgA κ mesangial cells and κ free light chains mesangial cells
Fig. 64: Presence of IgG κ mesangial cells and IgM κ mesangial cells
165
Fig. 65: Presence of 2 IgG κ mesangial cells, IgM κ mesangial cells
Fig. 66: Presence of κ free light chains mesangial cells
Fig. 67: Presence of λ free light chains mesangial cells
166
Fig. 68: The Bence Jones protein test
Fig. 69: Once the presence of the Bence Jones protein is demonstrated (in this case κ type) in the serum and in the urine it is obligatory to report not only the situation but also the dosage of the two mesangial cells separately
Fig. 70: The sample on left shows the presence of IgA λ mesangial cells. Furthermore, the presence of a confirmed type κ BJ is noted. The sample on right shows the presence of IgA λ mesangial cells. Furthermore, the presence of a confirmed λ BJ is noted
167
Fig. 71: The sample shows the presence of a confirmed λ type BJ in urinary immunofixation electrophoresis
Fig. 72: Presence of IgG κ mesangial cells and free light chains κ (?); the urinary immunofixation electrophoresis demonstrates the presence of the IgG κ mesangial cells and double κ (Bence Jones)
Fig. 73: On the left the sample shows the presence of IgG κ mesangial cells; the subsequent search for the presence of a proteinuria Bence Jones, on the right the urinary immunofixation electrophoresis shows the presence of IgG κ in the urine. In these cases it is good practice to use the antifree antiserums to verify if there is a monoclonal free light chain
168
Fig. 74: The sample on the left shows the presence of IgG λ mesangial cells, IgA λ mesangial cells and 2 IgM κ mesangial cells. The sample on the right shows the presence of IgG mesangial cells λ and IgM λ mesangial cells
Fig. 75: The sample on the left from hepatic transplant patient shows the presence of two IgG λ mesangial cells-at the α1 antitrypsin height, of a very weak IgG λ in position C3, of IgG heavy chain and two free light chains κ mesangial cells. The sample on the right has two IgG λ mesangial cells and IgM λ mesangial cells
Fig. 76: The sample on the left shows the presence of IgG λ mixed cryoglobulinemia and free light chains λ mixed cryoglobulinemia (positive Bence Jones) confirmed with antiserum antifree light chains. The sample on the right shows presence of IgA κ mixed cryoglobulinemia and a second free light chains κ mixed cryoglobulinemia (positive Bence Jones) confirmed with antiserum free light antichains
169
 
Cerebrospinal Fluid Oligoclonal
Analysis of oligoclonal bands gained from cerebrospinal fluid is shown in Figures 77 and 78.
Fig. 77: The sample shows the presence of oligoclonal pattern
Fig. 78: Cerebrospinal fluid with oiligoclonal pattern
 
Proteinuria
Diagnosis of proteinuria has been shown in Figures 79 to 90.170
Fig. 79: The various proteinurias
171
Fig. 80: A diagram of the different fractions of urinary protein of a glomerular and tubular origin
172
Fig. 81: The different fractions of tubular proteins Keywords: FLC, Free light chains; RBP, Retinol-binding protein.
Fig. 82: The different fractions of urinary glomerular proteins
173
Fig. 83: A sample of the different fractions of urinary protein of a glomerular and tubular origin
174
Figs 84A and B: Selective glomerular proteinuria
175
Figs 85A and B: Nonselective glomerular proteinuria
176
Figs 86A and B: Tubular proteinuria
177
Figs 87A and B: Mixed proteinuria
178
Figs 88A and B: Overloaded proteinuria with Bence Jones
179
Figs 89A and B: Overloaded proteinuria with lysozyme
180
Figs 90A and B: Postrenal proteinuria

CryoglobulinCHAPTER 6

There are some cases where there have been interpretive problems in the reading of the protein serum strip and/or the immunofixation. One typical example is represented by the presence of cryoglobulins (CR) in the serum. CR are proteins and their complexes that precipitate at low temperature and that generally dissolve again at 37°C. The phenomenon of the cryoprecipitability was studied and described for the first time by Heidelberg and Kendall in 1929 and then taken back and correlated with clinical manifestations of Wintrobe and Bull in 1933 in a woman carrier of myeloma. In 1947, Lerner and Watson first coined the term “cryoglobulins” referring to a group of immunoglobulins (Ig) that had the propriety to precipitate and to return, almost always, in solution if brought back to a temperature of 37°C.
 
HISTORY
In 1933, Winthrobe and Buell first observed the precipitation of protein upon cooling serum from a patient with Raynaud's phenomenon and the subsequent dissolution of the protein upon sample rewarming. In 1947, Lerner and Watson coined the term “cryoglobulin”. In the mid-1960s, Meltzer described the syndrome of purpura, arthralgia, asthenia and renal disease in association with CR and immune complex deposition. In 1974, Brouet et al. popularized a classification of CR according to their immunochemical composition. In the late 1980s, because chronic hepatitis, mainly hepatitis C (HCV), was frequently observed during the clinical course of mixed cryoglobulinemia (MC), a role for hepatotropic viruses in the pathogenesis of the condition was suggested and later confirmed. Based on this finding, many cases of MC, that in the past were called “essential cryoglobulinemia”, are now recognized as being due to viral infections, mainly HCV (Fig. 1).182
Fig. 1: Vasculitis and cryoglobulinemia related to hepatitis C
 
INTRODUCTION
Cryoglobulins are special serum Ig that precipitate at temperatures less than 37°C, but mostly at 0–4°C, and that dissolve when rewarmed to 37°C. The majority (95%) of CR are immune complexes (IC) that contain rheumatoid factor (RF). Such CR is known as “mixed” CR to differentiate them from the CR with monoclonal bands that do not contain RF or antigen antibody complexes. The antigens present in the mixed complexes may include RNA and proteins (e.g. hepatitis C virus proteins). A small fraction of CR (5%) comprises pure monoclonal gammopathies, which have poor solubility at low temperatures because of their unique amino acid sequences. There are other cryoproteins also that precipitate in the cold, such as cryofibrinogen (in plasma) and fibronectin.
Cryoglobuline analysis is an important test in the care of patients with vasculitis. However, in routine clinical laboratories and clinical practice, this test has not received adequate attention. This is due to several problems. Serum CRs in most of the patients exist in low concentrations (100–300 mg/L) among the high concentrations (60,000–80,000 mg/L) of normal serum proteins. It is difficult to isolate such small amounts of CR without contamination from normal serum proteins. CR, like other Ig, are variable in amino acid composition and are heterogeneous, they behave differently in vivo and in vitro. Analytical methods for CR have not kept up with recent advances in technology. For example, the sample volumes required for CR tests are large (5–10 ml of serum), the analysis is slow (3–7 days), and routine quantification techniques need refinement. Standards and controls are lacking. In addition, clinical laboratory personnel often lack experience in interpreting the electrophoretic patterns and the quantitative results of serum CR tests; they have scant appreciation of the importance of low CR levels.183
A wide gap exists between the knowledge of CR in research and in routine analysis. The aim of this chapter is to provide an overview of cryoglobulinemia and to focus attention on two areas: first, the importance of CR testing in patient care, especially for the low levels; and second, to suggest ways to improve the analytical techniques. The goal is to stimulate CR testing in routine patient care.
 
PATHOPHYSIOLOGY
The mechanisms of cryoprecipitation are poorly understood, but several factors have been investigated. The solubility of CR has been found to be partially related to the structure of component Ig heavy and light chains. Alteration in protein conformation with temperature changes also leads to decreased solubility and subsequent vasculitic damage. The ratio of antibody to antigen in circulating CR aggregates or IC affects the rate of clearance from the circulation and the resultant rate and location of tissue deposition.
Some cryoglobulinemia are thought to be related to immune complex disease (e.g. glomerulonephritis and chronic vasculitis), but not all persons with cryoglobulinemia present with these manifestations. Individuals with cryoglobulinemia may have intravascular CR deposits, a reduced level of complement, and complement fragments (C3a and C5a) that act as chemotactic mediators of inflammation; however, the pathophysiologic process of this disease has not been fully explained. Other sequelae are directly related to cryoprecipitation in vivo, including plugging and thrombosis of small arteries and capillaries in the extremities (gangrene) and glomeruli (acute renal failure). Circulating large molecular weight cryoprotein complexes, even when unprecipitated in vivo, can lead to clinical hyperviscosity syndrome.
Type I cryoglobulins are usually monoclonal Ig (IgM) and, less frequently, IgG, IgA or light chains. Type I cryoglobulins rarely have RF activity and do not activate complement in vitro. This disorder is typically related to an underlying lymphoproliferative disease and, as such, may be clinically indistinguishable from Waldenström macroglobulinemia, multiple myeloma or chronic lymphocytic leukemia. Type I cryoglobulinemia may result in hyperviscosity due to high levels of circulating monoclonal CR, leading to physical obstruction of vessels. Concentrations may reach up to 8 g/L. In addition, non-obstructive damage may be mediated by immune complex deposition and subsequent inflammatory vasculitis (Figs 2A and B, Table 1).
Types II and III cryoglobulins, also known as the MC, are associated with chronic inflammatory states, such as systemic lupus erythematosus (SLE), Sjögren syndrome and viral infections (particularly HCV). In these disorders, the IgG fraction is always polyclonal with either monoclonal (type II) or polyclonal (type III) IgM (rarely IgA or IgG) with RF activity (ability to bind IgG). B cell clonal expansion, particularly RF secreting cells, is a distinctive feature in many of these disease states (Table 1). 184
Table 1   Disorders associated with the different types of CR
Type I
  Monoclonal gammopathy
  Multiple myeloma
  WM
Type II
  Hepatitis C virus
  Autoimmune disease
  Sjögren's syndrome
  Chronic lymphocytic leukemia
  Non-Hodgkin's lymphoma
Type III
  Systemic lupus erythematosus (SLE)
  Biliary cirrhosis
  Rheumatoid arthritis
  Viral infections (CMV, Epstein Barr, HIV, HBV)
  Bacterial infections (leprosy, spirochetes)
Figs 2A and B: (A) Gangrene and type I cryoglobulinemia; (B) Mixed cryoglobulinemia
The resultant aggregates and IC are thought to outstrip reticuloendothelial clearing activity. Tissue damage results from immune complex deposition and complement activation. Of note, in HCV related disease, HCV related proteins are thought to play a direct role in pathogenesis and are present in damaged skin (Figs 2A and B), blood vessels and kidneys.185
Table 2   Classification of CRs
Type
Composition
Description
I
Isolated IgM
These are composed of a single monoclonal immunoglobulin paraprotein (usually IgM). Sometimes, these are represented by light chains only and can be extracted from the urine, or they will accumulate in blood serum in the event of renal failure
II
Immunocomplexes formed by IgM
They usually have a polyclonal component, usually IgG, and a monoclonal component, usually IgM or IgA, which has an RF function. The IgM can recognize intact IgG or either the Fab region or Fc region of IgG fragments. This is why most type II CRs are IgM-IgG complexes
III
Immunocomplexes formed by polyclonal IgM
These have very similar function to the type II CRs; however, they are composed of polyclonal IgM and IgG molecules
 
CLASSIFICATION
Cryoglobulinemia is classically grouped into three types according to the Brouet classification (Table 2). Type I is most commonly encountered in patients with a plasma cell dyscrasia, such as multiple myeloma or Waldenström macroglobulinemia and requires special and more aggressive treatment. Type I, although not common, can be recognized easily. It is characterized by high serum CR concentration (> 2 g/L) with clearly visible turbidity or precipitate. The CR precipitates (or gels) easily and rapidly, sometimes at room temperature. CR may be detected incidentally in the laboratory because of interference in other laboratory tests. Electrophoresis confirms the presence in the serum of a large monoclonal protein band that is also present in the precipitate, but at a higher proportion (relative to albumin) when compared to that in the serum. Immunofixation is used to identify the type of the monoclonal protein for diagnosis and treatment (e.g. WM, which causes high serum viscosity, or multiple myeloma, which represents malignancy). From a clinical perspective, any patient with monoclonal gammopathy and cold sensitive symptoms should be examined for CR.
In cryoglobulinemias of types II and III, the warm serum does not contain any monoclonal peak. In type II, the CR precipitate contains only a monoclonal peak, whereas in type III no monoclonal peak is present in the CR precipitate. Differentiating type II from type III has less immediate importance since both types indicate immunostimulation and/or infection. However, differentiating types II and III is important for prognosis, since type II may eventually develop into non-Hodgkin's lymphoma. The serum CR concentration is usually very high in type I, so from CR quantitation a good idea about the typing can be deduced.186
 
Cryoglobulinemia Ist Type Sec. Brouet
  • Macro aggregated for intramolecular electrostatic interactions
  • IgM that has the tendency to form with itself complexes
  • Greater frequency for IgM κ
  • Lesser frequency for IgG and subclasses IgG1, IgG2 and IgG3
  • Rarely IgA
Organization of the cryoprecipitate: Amorphous or without precise structure, tubular organized and hydrated, very compact crystalline (organization most damaging IgG3 in microcrystals).
 
Cryoglobulinemia IInd Type “a” Sec. Brouet
  • Immunocomplexes
  • The formative genesis show biological activity by Ig
  • IgM with RF activity against the IgG polyclonals (rarely against IgA)
  • Tendency to form complexes with polyclonal Ig and RF
  • The RF acts in particular on the aggregated Ig and less on the natives, the RFs arise from abnormal proliferation of a plasma cellular clone
  • A single monoclonal band to the immunofixation.
 
Cryoglobulinemia IInd Type “b” Sec. Brouet
  • Immunocomplexes
  • The formative genesis show biological activity by Ig
  • Igs oligoclonals (IgM) with RF activities against IgG polyclonals (rarely against IgA)
  • Tendency to form complexes with IgG polyclonals and RF
  • RF acts in particular on the Ig aggregates and less on the natives. The RFs arise from abnormal proliferation of a plasma cellular clone
  • Two monoclonal bands to the immunofixation.
 
Cryoglobulinemia of IIIrd Type Sec. Brouet
  • Ig polyclonals
  • Composition derived by one or more Ig.
The RFs arise from disturbances and magnification of the phlogiston process of immunomodulation, during the response to the antigen. Naturally, as happens in many situations, there are forms currently recognized by Musset (1992), Bellotti (1991), that are not within the framework of the above cited classes, leading us to hope for a more simplified classification and limited to two classes in which the first class we have the monoclonal proteins and in the second, all the forms of mixed type.
 
“Microheterogene” Globulins Sec. Musset
  • Presence of two or more, light, monoclonal components
  • Polyclonal context present
  • Greater frequency of IgG and IgM.
In confirmation of what is proposed, taking into account various works presented in literature, there is homogeneity in terms of the incidence of 187Ist type CR according to Brouet and what other authors define as monoclonal type cryoglobulinemia, while there is extremely inhomogeneous data for other forms, furthermore, highlighting heterogeneity in the selection of the Cryoglobulinemic population and a lack of standardization of the same methodologies.
Fig. 3: The three types of cryoglobulins as detected by capillary electrophoresis (C, cryoglobulin; S, serum; A, albumin; M, monoclonal band; G, globulins)
Types II and III have RF activity and bind to polyclonal Ig. These two types are referred to as MC. When the temperature is raised, the precipitated CRs will dissolve back into the serum. In 2006, it was discovered that there are unusual CRs that show a microheterogeneous composition with an immunochemical structure that cannot be fit into any of the classifications. A classification of a type II—III variant has been proposed, because they are composed of oligoclonal IgMs with traces of polyclonal Ig (Fig. 3).
 
Cryoprecipitate Composition
Several components can be found in CR precipitates in addition to Ig, including RF, albumin, fibronectin, fibrinogen, viruses and bacteria. Some of these are contaminants or co-precipitants (e.g. albumin and the normal Ig). In CR type I only an IgM should be detected. However, in the mixed CR (types II and III) many components can be detected including RF, which is composed of IgM-K (monoclonal in type II and polyclonal in type III). Because these complexes have RF activity they can cause immune complex vasculitis 188in target organs, such as skin, nerves, kidneys, liver, and joints. The RF is complexed with polyclonal Ig directed against the stimulating agent (viruses, bacteria or specific immunogens). By itself, the RF does not precipitate in the cold, but it does so when bound to polyclonal or IgM.
 
Cryoprecipitation Mechanism
The solubility of proteins depends on many factors, such as the protein concentration, hydrophobicity, size and surface charge, as well as the solution temperature, pH and ionic strength. Changes in the primary structure of the variable portion of Ig, and reduced concentrations of sialic acid and galactose in the Fc region of Ig, may be related to decreased solubility of CR. Because of the high molecular weight of CR complexes (i.e. mixed CR), they are less soluble and tend to precipitate at cold temperatures in vitro and in vivo. Production of IgM-RF complexes represents a crucial factor in the pathophysiology of cryoprecipitation. The formation of cryoprecipitation aggregates upon exposure to cold may be the triggering factor for vasculitis; however, this does not explain why tissues (e.g. kidney and nerves) that are distant from the site of exposure to cold may be affected. One explanation, suggested by in vitro studies, is that alterations of chloride concentration may influence CR structure and aggregation.
 
Rheumatoid Factor
Low affinity RFs (often K light chains) are natural poly reactive antibodies present in human serum that have specificity for IgG. The normal role of RF in the immune response is unclear, but it probably aids in immune complex clearance by making complexes larger and activating complement. The RF cross reacts with other autoantigens and binds to microorganisms covered with specific IgG antibodies, leading to agglutination and complement activation. It is postulated that HCV infects circulating B lymphocytes, stimulating them initially to synthesize polyclonal IgM RF. However, unknown factors induce a shift to abnormal proliferation of a single clone of B cells that produces IgM-K RF, leading to type II MC. As have been discussed later, CR similar to those in the serum can be detected in white cells of the patients. HCV RNA is found concentrated and bound to the CR precipitate. Patients with HCV and cryoglobulinemia type II have been found to have a Bcl 2 rearrangement in peripheral blood leukocytes. Occasionally, a small monoclonal protein (IgM RF type II CR) is present in the plasma of hepatitis C patients, which resembles the small paraprotein in monoclonal gammopathy of undetermined significance. IgM-K RF binds avidly to anti-HCV IgG or to the IgG-HCV immune complex leading to the presence of CR in the serum. These circulating IC concentrate in capillaries of different tissues (e.g. renal glomeruli), where they deposit in the subendothelium and initiate cellular proliferation and leukocyte infiltration. Chronic HCV infection may also produce autoantibodies to native renal antigens, which may account for some of the glomerular pathologic response that occurs in membranous glomerulonephritis. Elevated levels of RF seem to be associated with higher incidence of CR. In many patients with type II cryoglobulinemia, the CR is 189not simply monoclonal RF, but an oligoclonal form. That is termed type II–III. This may represent a transition from type III to type II.
 
Clinical Manifestations
The common symptoms of CR are due to cutaneous ischemia, and include purpura, livedo reticularis, ecchymosis, ulcerations, ischemic necrosis, and, rarely, gangrene. Other manifestations of CR are chronic hepatitis, membranoproliferative glomerulonephritis, vasculitic neuropathy, dysesthesia, Raynaud's syndrome and secondary Sjögren's syndrome. In one study, the typical clinical triad purpura, weakness and arthralgia were present in almost 80% of patients at the time of diagnosis. Purpura remains the main (≍80%) clinical sign of cryoglobulinemia. It is intermittent, with palpable lesions appearing on the lower limbs and less frequently on the buttocks, trunk or face. Lower limb purpura is usually preceded by paresthesia or local pricking sensations rather than frank pain. The eruptions vary greatly in frequency.
About 50–70% of symptomatic patients with cryoglobulinemia have liver involvement, arthralgia and asthenia, and about 25% have renal involvement. CR vasculitis affects the kidney usually with a membranoproliferative glomerulonephritis and affects the prognosis and the survival rate of the patients. The incidence of nervous system involvement is 36%, affecting the peripheral nervous system and presenting as sensory motor neuropathy, especially in the lower limbs, with painful paresthesias and loss of strength.
 
CR Concentration and the Severity of Symptoms
Normal persons have very low serum concentrations of CR (0–60 mg/L). Because CRs are heterogeneous compounds that vary in chemical composition, typing, thermal properties, and ability to stimulate complement, should not be expected that their serum levels would correlate well with the severity of symptoms.
Immune mediated vasculitic lesions are responsible for the different clinical symptoms of cryoglobulinemia, including cutaneous and visceral organ involvement. CR of types II and III are accompanied by RF, activate complement and thereby cause vasculitis. Type III is not associated with development of malignancy and is less associated with vasculitis. Type II occurs with chronic infections, but it can lead, as discussed later, to leukemia. Since type I CR is not accompanied by RF or IC, this type does not activate complement and causes less vasculitis but more hyperviscosity. Type I is associated with B cell malignancy. Thus, the different CR act through different and complex mechanisms.
When HCV patients with cryoglobulinemia are compared as a group to those without cryoglobulinemia, the severity of disease is higher in the CR positive group. For example, patients with CR had increased fibrosis and increased incidence of cirrhosis compared to those without CR, but there was no significant correlation between the serum CR level and hepatic histological activity index. Also, in patients with mixed cryoglobulinemic 190glomerulonephritis, correlation between serum CR level and disease activity was not significant. Similar findings were reported for the development of lymphoma in steroid dependent patients. The fact that many patients have cryoglobulinemia without symptoms, even when serum CR levels are high, indicates that clinical symptoms and severity do not depend simply on the serum CR concentration but on other factors mentioned above.
Group of scientists observed that “there is an inverse relation between vasculitis and the cryocrit—the lower the cryocrit, the greater the frequency of signs of vasculitis, especially in type II MC. This is a still unexplained phenomenon, but it seems to be related to the intrinsic capacity of IC to activate complement in situ.” Because of the lack of correlation between serum CR concentration and severity of disease, low CR values should not be ignored. Sensitive methods for detection CR at low levels are important for patient diagnosis and prognosis. Furthermore, in patients with a positive CR test, the CR level is useful for following the response to treatment.
 
Laboratory Studies
Cryoglobulin assays have not been adequately standardized. The precipitation step of CR is often incomplete, especially for type III. CR precipitates are generally contaminated with normal serum proteins. The presence of albumin as measured by electrophoresis or microalbumin assay confirms this fact. Most contamination of the CR precipitate comes from serum proteins that adhere to the walls of the tube. Washing steps can decrease the serum protein contamination, but they also cause a loss of CR precipitate, especially when dealing with small volumes. It is wise to minimize the washing steps and to correct for serum protein contamination based on microalbumin assay.
Cryoglobulins vary substantially from one patient to another. CR differs widely in the temperature and rate of precipitation (type I can precipitate in few minutes while type III may require a week). Some CR dissolves easily in warm saline while others are difficult to redissolve. The majority of CR is types II and III that are unstable when frozen. They should be analyzed promptly. On the other hand, some CR samples become denatured and precipitate if left for a few days at room temperature. Redissolution of the CR precipitate upon rewarming is important for CR confirmation. The differences in prevalence of CR in various studies may reflect differences in analytical sensitivity and techniques.
Several methods have been described for CR analysis, including electrophoretic and nonelectrophoretic methods (e.g. cryocrit assays, immunoassays, protein assays by ultraviolet absorption, etc.). The electrophoretic methods have the advantage that they do not need sample washing to remove non CR serum proteins. The results can be corrected for serum protein contamination by comparing the ratio of albumin/Ig in the CR precipitate to that in the serum. The electrophoretic methods also allow direct phenotyping. Most methods for CR quantification ignore the fact that the cryoprecipitate is heavily contaminated by normal serum proteins, as evidenced by the presence of albumin, unless the precipitate is washed repeatedly. Measurement of RF has been suggested instead of CR.191
In type I cryoglobulinemia, which is relatively rare, the blood sample should be collected and brought to the laboratory at 37°C. However, in patients with mixed CR (types II and III), blood samples can be collected and transported to the laboratory at room temperature.
 
Visual Screening and Qualitative Detection of CR
Cryoglobulins cause serum to become turbid upon cooling. Visual observation of turbidity is the easiest method of detecting CR. However, the turbidity is neither specific nor quantitative, especially at low levels where lipids (especially chylomicrons) and fibrinogen may interfere. Visual inspection of the CR precipitate after centrifugation (preferably in the cold as a cryocrit) avoids these problems and is more sensitive, especially when a portion of the serum sample is kept at 37°C as a control. Different CR produces different degrees of turbidity. In about half of the positive samples, serum CR concentrations are less then 300 mg/L and such levels are difficult to detect visually based on turbidity. However, CR levels close to the upper reference range (≍100 mg/L) can be detected after centrifugation of the cold and warm tubes.
 
Semiquantitative Detection of CR by Cryocrit
The cryocrit is a simple and widely used method. A special conical tube (Wintrobe) is filled with 5–10 mL of serum and left in a refrigerator for 3–7 days. The tube is centrifuged and the volume of the CR precipitate is read from the tube markings. The precipitate can then be washed and analyzed further. This method has good sensitivity, but quantification of the CR precipitate as a cryocrit requires a large volume of serum, is affected by the speed of centrifugation, and assumes that different proteins sediment with equal volumes.
 
Colorimetric Assay of CR
Serum (1 ml) in a conical centrifuge tube (similar to the cryocrit tube) is kept in an ice water bath for 3 days and centrifuged for 5 minutes at 5,000 rpm. The top layer is discarded. The tube is gently washed 3 times with 3 ml of ice cold water, without disturbing the precipitate, followed by centrifuging for 2 minutes. The CR precipitate is dissolved in 1 ml of warm (37°C) saline solution (1%, w/v). The CR precipitate is measured by a urine protein assay (e.g. pyrogallol red or Coomassie blue) and its contamination by albumin is measured by a urine microalbumin immunoassay. The albumin content is used to correct for contamination of the CR precipitate by normal serum proteins. However, if the albumin concentration is more than 100 mg/L, the correction is unreliable (Figs 4A to C.
 
Electrophoretic Phenotyping and Quantification of CR
Electrophoretic methods quantify and phenotype CR at the same time. We have used both gel and capillary electrophoresis (CE) for CR quantification and typing. The CE procedure is more rapid and accurate than gel 192electrophoresis for CR quantification.
Figs 4A to C: (A) Normal human serum negative CR; (B) Cryoprecipitate including a mixed type II CR (IgM-κ and polyclonal IgG); (C) Type I with complete serum gelification
The accuracy stems from avoiding dyes for measuring proteins and basing the calculation on ratio measurements. The CR precipitate from the cryocrit or colorimetric assay is washed only twice and subjected to electrophoresis. The increase of the Ig/albumin ratio in the CR precipitate, relative to that in the serum, is used to calculate the CR. The reference range for serum CR concentration by the CE method is 0–60 mg/L.
 
Other Methods
Other quantification methods for CR have been described but have not gained wide acceptance. These include laser nephelometry diffusion of CR in gels, and light scattering, for type I cryoglobulinemia, immunofixation analysis is used to determine if the CR is due to multiple myeloma or WM. For cryoglobulinemia of types II and III, the immunofixation technique is not helpful.
 
Procedure for Testing Serum CR
  • The blood specimen must be obtained in warm tubes (37°C) in the absence of anticoagulants
  • Allow the blood sample to clot before removal of serum with centrifugation (at 37°C)
  • The period required for the serum sample to incubate (at 4°C) depends on the type of CR present, as follows:
    • Type I tends to precipitate within the first 24 hours (at concentrations >5 mg/mL)
    • Type III CRs may require 7 days to precipitate a small sample (<1 mg/mL) (Fig. 5)
  • Repeat centrifugation to determine cryocrit (volume of precipitate as a percentage of original serum volume)193
Fig. 5: Procedure for testing serum CR
  • Cryoglobulin concentration may be determined via spectrophotometric analysis. Specific immunologic assays may be used to identify CR components (Ig, light chains, clonality)
  • Urinalysis: Abnormalities may represent evidence of renal disease
  • Complete blood cell count: Leukocytosis may be a manifestation of concomitant infection or leukemia. Anemia may be present
  • Serum chemistry: Patients with renal insufficiency may present with elevated serum creatinine levels and electrolyte abnormalities
  • Liver function studies: Liver function studies may reveal evidence of underlying hepatitis; obtain hepatitis serology
  • RF: RF is positive in types II and III
  • Antinuclear antibody (ANA): ANA is indicated upon clinical suspicion of underlying connective tissue disease (SLE, Sjögren syndrome)
  • Erythrocyte sedimentation rate (ESR): Elevations may be secondary to Rouleaux formation194
  • Complement evaluation (CH50, C3 and C4): Patients may display hypocomplementemia (especially low C4 levels)
  • Other studies: Consider serum protein electrophoresis (SPE), urine protein electrophoresis (UPEP) and quantitative Ig upon suspicion for underlying gammopathy
  • Serum viscosity: Measure serum viscosity if symptoms warrant
  • Further diagnostic laboratory tests: Consider further testing based on the level of suspicion for other associated disease. For example, a recent study demonstrated that patients with MC associated with HCV infection have elevated levels.
 
Histologic Findings
Skin: Purpura is histologically characterized by dermal vasculitis that extends variably to the subcutaneous interstitial space. HCV associated proteins have been found in vasculitic skin biopsy samples, suggesting a role for these antigens in pathogenesis of the lesions.
Other organs: Autopsy studies have revealed unsuspected vasculitis of multiple organs (heart, lung, gastrointestinal tract, central nervous system, liver, muscle, adrenals, etc.). Histologic evaluation of affected lung, kidney and muscle reveals eosinophilic material in the lumen of small vessels with frequent extension into the vessel intima and inflammation of the vessel wall.
Although biopsy samples generally exhibit inflammatory vascular changes (e.g. leukocytoclastic vasculitis in patients with vasculitic purpura), intraluminal CR deposits may be observed, especially in renal glomeruli (Fig. 6).
 
Reporting Results of CR Tests
Negative results are reported as such. When a serum sample is positive for CR, the concentration and phenotype of serum CR are reported, as well as the concentration of total γ globulin. Since serum RF assays are positive in a majority of cryoglobulinemic patients and the concentrations of RF correlate with CR levels, serum RF concentration is routinely analyzed and the results are included.
Figs 6A and B: A. Renal biopsy sample that shows membranoproliferative glomerulonephritis in a patient with hepatitis C-associated cryoglobulinemia (hematoxylin and eosin; magnified X200); B. An electron micrograph of a kidney biopsy specimen from a patient with cryoglobulinemia
195
 
Interferences in CR Analysis
Hemoglobin precipitates as CR and interferes in the analysis. In electrophoresis, hemoglobin can be detected as a sharp peak that migrates ahead of the Ig. Hemolyzed samples should not be tested for CR unless they are to be analyzed by electrophoresis. Fibrinogen and heparin also interfere. It is difficult to solubilize fibrinogen, but the presence of fibrinogen in a precipitate can be confirmed by immunoassay. As discussed above, the main interference in CR assays is the contamination from normal serum Ig.
 
Interference of CR in Other Tests
Serum samples that contain CR sometimes gel or precipitate and clog analytical instruments. CR that precipitates during serum centrifugation or storage may elude detection; upon electrophoresis, such samples can exhibit a normal serum pattern despite having a large monoclonal protein. The CR may precipitate at the application point on the electrophoretic membrane. When a CR precipitate traps other Ig, they may confuse the interpretation of the immunofixation pattern. The presence of CR can cause false elevated cell counts in automated cell counters.
 
Interpretation of the Results
Normally, there are no CRs. Negative CR test found in most healthy people and is not routinely ordered on those without symptoms. When the test is positive, it means that CR is present and has the potential to precipitate upon exposure to cold. The symptoms experienced when this happens will vary from person to person, may be different with each exposure, and will not necessarily correlate with the quantity of CR present (Figs 7 to 14).
Fig. 7: IgG-κ monoclonal type I CR
Fig. 8: IgM-κ monoclonal type I CR
196
Fig. 9: IgG-κ monoclonal and a low concentration of IgG polyclonal type IIa CR
Fig. 10: IgM-κ monoclonal and a low concentration of IgG polyclonal type IIa CR (hepatitis C sample)
Fig. 11: IgG-κ monoclonal, IgA λ monoclonal and a low concentration of IgG and IgM polyclonal type IIb CR (hepatitis C sample). SPE line shows a β lipoprotein band
Fig. 12: IgG and IgM oligoclonal with Igs polyclonal type IIb CR (hepatitis C sample). SPE line shows a β lipoprotein band
 
TREATMENT
The treatment of cryoglobulinemia depends on the severity of symptoms, the underlying disease, and the type of CR. The goal of therapy is to limit in vivo precipitation of CRs and the resultant inflammatory effects. Asymptomatic patients usually do not need treatment.197
Fig. 13: IgG and IgM polyclonal type III CR
Fig. 14: Cryofibrinogen immunofixation
Even in the presence of high cryocrit levels. Some patients with cryoglobulinemia suffer from mild, recurrent episodes of lower extremity purpura that require no specific therapy or only therapy with general anti-inflammatory drugs (e.g. ibuprofen, aspirin) or low doses of steroids. When there is evidence of organ involvement such as vasculitis, renal disease, progressive neurological findings, or disabling skin manifestations, especially in absence of HCV, cryoglobulinemia is treated by suppression of the immune response (e.g. corticosteroid therapy, cyclophosphamide and azathioprine). In all cases, careful clinical monitoring of the disease, with particular attention to severe vasculitic or neoplastic complications, is mandatory.
In multiple myeloma with type I CR, cytotoxic drugs (e.g. melphalan, prednisone, cytoxan, chlorambucil) are prescribed. Approximately one half of myeloma patients respond to biaxin with more than 50% reduction in monoclonal component, a response that is comparable to standard therapy with melphalan and prednisone. Plasmapheresis is indicated for severe complications related to cryoprecipitation or serum hyperviscosity syndrome. It removes circulating CRs and prevents their deposition in glomeruli. Thalidomide has also been used to treat type I cryoglobulinemia.
Acute nephritic or nephrotic flare ups with rapid deterioration of renal function and systemic vasculitic episodes associated with type II CR are 198treated with corticosteroids and plasmapheresis. Plasmapheresis alone is not very effective because although it removes circulating CRs, it does not suppress their production. Extensive vasculitis may respond to prednisone, cyclophosphamide, or both. Some have recommended concomitant cytotoxic medications and corticosteroids to reduce the rebound of CR phenomena that may develop after plasmapheresis.
Interferon α (IFN) and PEG interferon α have been effective in patients with cryoglobulinemia associated with hepatitis C and in patients with chronic myelogenous leukemias and low grade lymphomas. However, the effects are temporary, and relapse may occur within a few months after discontinuation of the treatment. IFN is contraindicated in acute flare ups, since its immunostimulatory activity may aggravate acute flares of acute nephritis, nephrotic syndrome, and systemic vasculitis. In the presence of acute cryoglobulinemic GN, IFN does not prevent the progression of renal damage. Combination therapy with cytotoxic and anti-inflammatory drugs, and sometimes plasma exchange, has been shown to improve renal function. A study by Sarac and colleagues demonstrated that high doses of IFN (10 million units, 3 times/week) resulted in sustained improvement of renal disease in a few patients. This regimen may induce remission in patients who fail to respond to conventional doses of IFN.
In patients with chronic hepatitis C without renal involvement, combination therapy with IFN and ribavirin (Rebetron) has been shown to be superior to IFN alone in inducing longer remission and may eradicate HCV infection in a considerable number of subjects. Patients in whom antiviral therapy is ineffective, contraindicated, or not tolerated, rituximab, a monoclonal chimeric antibody that binds to the B cell surface antigen CD20 with selective B cell blockade, may be a useful alternative to standard immunosuppression.
In type III CR the treatment is generally with anti-inflammatory drugs (e.g. ibuprofen, aspirin) and steroids.

Lipoprotein ElectrophoresisCHAPTER 7

 
INTRODUCTION
Lipoproteins provide a means for fat transport between different organs and tissues. Their main clinical importance is related to their role in the development of atherosclerosis. The risk of atherosclerosis is increased in noninsulin-dependent diabetes or type 2 diabetes mellitus that is known to be associated with raised plasma lipids. The control of two enzymes, hormone sensitive lipase (Ec 3.1.1.3; HSL) and lipoprotein lipase (Ec 3.1.1.34; LPL) during fasting and feeding is thought to be important for the maintenance of low plasma triglyceride (TG) and fatty acid levels, which reduce the risk of heart disease. Hormone sensitive lipase is the intracellular enzyme responsible for the hydrolysis of stored TG during the process of lipid mobilization from adipose tissue. Lipoprotein lipase controls lipid hydrolysis for the clearance of TG from circulating lipoprotein particles in the plasma.
 
LIPOPROTEIN METABOLISM
The lipoprotein system evolved to solve the problem of transporting fats around the body in the aqueous environment of the plasma. A lipoprotein is a complex spherical structure, which has a hydrophobic core covered by a hydrophilic coating. The core contains TG and cholesteryl esters, while the surface contains phospholipid, free cholesterol, and proteins. The proteins associated with lipoproteins are referred to as apolipoproteins (Apos) or apoproteins. Some of their properties are given in Table 1. Cholesterol is an essential component of all cell membranes and is the precursor for steroid hormone and bile acid biosynthesis. There are several different classes of lipoproteins whose structure and functions are closely related.200
Table 5.1   Properties of human apolipoproteins
Apolipoprotein
Molecular
weight (kD)
Site of synthesis
Function
A I
28
Intestine, liver
Activates LCAT
A II
17
Intestine, liver
  
B 100
549
Liver
• TG and cholesterol transport
• Binds to LDL receptor
B 48
264
Intestine
TG transport
C I
6.6
Liver
Activates LCAT
C II
8.85
Liver
Activates LPL
C III
8.8
Liver
Inhibits LPL
E
34
Liver, intestine,
macrophage
Binds to LDL receptor and probably also to another specific liver receptor
LCAT, lecithin cholesterol acyltransferase; LDL, low density lipoproteins; LPL, lipoprotein lipase
Lipoproteins can be classified according to their hydrated density into chylomicrons (CM), very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). With decreasing TG content, the density of the lipoprotein particle increases and the size decreases, from CM through VLDL, IDL, LDL to HDL, which are the smallest. Lipoproteins can also be classified by their electrophoretic mobility, where α-lipoproteins (HDL) migrate furthest, followed by pre-β-lipoproteins (VLDL) and β-lipoproteins (LDL). Chylomicrons remain at the origin of the electrophoretic strip as cited by Dominiczak (1999). The metabolism of lipoproteins can be divided into two pathways, depending on whether the lipid is of exogenous or endogenous origin (Figs 1A and B).
 
Exogenous Pathway
In the exogenous pathway, illustrated in Figure 1A, CM represents the major means of dietary lipid transport. Dietary TG is first hydrolyzed by intestinal pancreatic lipase and is absorbed as monoglyceride, nonesterified fatty acid (NEFA) and glycerol as cited by Dominiczak (1999). In the enterocytes, TG is synthesized, and together with phospholipids and cholesterol, is combined with Apo B 48 to form CM. CM are secreted into the lymph via the thoracic duct and reach the plasma where they obtain Apo E and Apo C I, C II and C III from HDL particles. CM normally appears in plasma after fat-containing meals. In the peripheral tissues, such as muscle and adipose tissue, CM is acted upon by LPL, which hydrolyses their TG component.201
Figs 1A and B: Major pathway of lipoprotein metabolism. A. The exogenous pathway; B. The endogenous pathway. The dashed lines show that particles may pass through several cycles of hydrolysis by LPL in capillary beds
HDL, high density lipoprotein; Apo, apolipoprotein; CM, chylomicrons; FA, fatty acids; LPL, lipoprotein lipase; LDL, low density lipoprotein; VLDL, very low density lipoprotein
202
As a consequence, the particles become smaller and are now called CM remnants. The remnants are rapidly removed from the circulation by hepatic receptors that are internalized in the liver by Apo E receptor-mediated endocytosis and degraded in lysosomes, thus delivering dietary TG and cholesterol to the liver. The half-life of CM in plasma has been estimated to be 5 minutes. The CM is lighter than water and will float to the top of the plasma when it is stored overnight in the refrigerator. Since plasma from a fasting specimen should not contain CM, the observation of this floating layer is significant and indicates a deficiency of peripheral lipase activity. CM is found in the fasting plasma of persons with type I and type V hyperlipoproteinemia.
 
Endogenous Pathway
 
Very Low Density Lipoprotein Metabolism
Very low density lipoprotein particles are synthesized and secreted by the liver and contain endogenously synthesized TG and cholesterol (Fig. 1B). Apo B 100 is an essential component. Apo C and E are incorporated into VLDL after secretion by transfer from HDL particles. As the particles circulate in the plasma, they are delipidated by the action of LPL in the same way as CM. The depleted particles called IDL particles, which may bind to the hepatocytes through Apo E receptors or have further TG removed producing LDL particles. By this stage all the Apos, except for Apo B 100, have been transferred from the particles to HDL. IDL or LDL particles can bind to receptors on hepatocytes where hepatic lipase (HL) causes further hydrolysis of TG, which may be necessary prior to their uptake. IDL is not found in significant amounts in the circulation unless there is a defect in conversion of VLDL to LDL. Such cases are caused by a deficiency of Apo E III or Apo C III activated lipase. This results in the accumulation of IDL in the plasma. This is responsible for type III hyperlipoproteinemia.
Excessively elevated VLDL is responsible for type IV hyperlipoproteinemia and is most often caused by hyperinsulinemia, which promotes TG production. When both CM and VLDL are greatly increased, the abnormality is defined as Type V hyperlipoproteinemia.
 
Low Density Lipoprotein Metabolism
Low density lipoprotein particles are cholesteryl ester enriched and their function is to deliver cholesterol to cells. They are formed from VLDL via IDL. They can pass through the junctions between capillary endothelial cells and attach to LDL receptors on cell membranes that recognize Apo B 100. Macrophages internalize LDL in endocytotic vesicles, which are passed on to lysosomes where the cholesterol esters are degraded by an acid hydrolase. This liberates free cholesterol, which leaves the lysosome and is either excreted or transferred to HDL or is re-esterified in the cytoplasm by acyl cholesterol acyltransferase (ACAT). Stored cholesterol ester can be hydrolyzed subsequently by a cytoplasmic neutral cholesterol ester hydrolase and passed to HDL, or in the absence of HDL, the free cholesterol is retained in the cell and re-esterified by ACAT. About 50% of the stored cholesterol 203esters are hydrolyzed and re-esterified each day via this cycle. The release of free cholesterol is responsible for three regulatory responses that assist in cholesterol homeostasis: (i) cholesterol synthesis is decreased primarily by suppression of the rate limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase; (ii) intracellular cholesterol storage is increased by activation of ACAT activity to esterify excess cholesterol; (iii) modulation of a number of LDL receptors on the plasma membrane to prevent over accumulation of intracellular cholesterol through the receptor pathway.
This is responsible for type II hyperlipoproteinemia. Low levels of LDL occur in two inherited conditions. Abetalipoproteinemia results from a complete deficiency of Apo B. This is an autosomal recessive condition associated with severe metabolic problems, including intestinal malabsorption, motor nerve dysfunction, fat soluble vitamin deficiency, and anemia.
 
High Density Lipoprotein Metabolism
High density lipoprotein metabolism is involved in cholesterol transport from the tissues to the liver for export from the body. High density lipoproteins are synthesized primarily in the liver and, to lesser extent, in small intestinal cells as disk-shaped precursor particles (nascent HDL). A major function of HDL is to act as a receptacle for excess phospholipid and cholesterol derived from cells or as by-products of lipolysis. The lipolysis of CM and VLDL by LPL results in a net transfer of phospholipid into HDL. Free cholesterol diffuses into HDL from other lipoprotein particles, erythrocytes and endothelial cells. The HDL-associated enzyme lecithin cholesterol acyltransferase (LCAT) uses phospholipid and cholesterol to generate cholesteryl esters and lysophospholipid, thus, driving the influx of further phospholipid and cholesterol into spherical HDL. The spherical HDL is relatively large cholesteryl ester-enriched particles known as HDL2. Their cholesteryl ester content can be transferred to remnant particles in exchange for TG, the process being mediated by cholesteryl ester transfer protein (CETP), a 74 kD protein, which is synthesized in the liver. Some of the cholesteryl ester from HDL2 particles is also transferred to the TG-rich lipoproteins. The resulting smaller HDL particles are known as HDL3 and are ready to accept further cholesterol from peripheral tissues. Thus cholesterol is transferred from peripheral tissues to the liver, from where it can be excreted as cholesterol and as bile salts in the bile. This process of removal of cholesterol from the tissues to the liver and ultimate excretion from the body is known as reverse cholesterol transport.
Absent or nearly absent HDL occurs in an autosomal recessive hypolipoproteinemia called Tangier's disease. This is caused by a deficiency of both Apo A I and Apo A II, the principle lipoproteins of HDL. Persons with this disease develop premature coronary artery disease (CAD).
 
Lipoprotein (a) or Lp (a)
Lipoprotein (a) contains Apo B bound to another apoprotein that is designated Apo (a). Like LDL, it is about 27% protein and 65% lipid by weight 204and has pre-β-mobility on electrophoresis. The amount of LP (a) in the plasma is normally below 150 mg/dL. Elevated levels are considered to be an independent risk factor for developing CAD. High levels are inherited as an autosomal dominant trait and are not influenced by diet or exercise. It is speculated that the link between Lp (a), and atherosclerosis is related to the similarity between Apo (a) and plasminogen. Plasminogen is the precursor of plasmin, which initiates the lysis of blood clots.
 
Lipoprotein X
Lipoprotein X is an abnormal lipoprotein often seen in patients with obstructive liver disease. It consists of unesterified cholesterol, phospholipids and protein. It migrates slower than LDL. Because of its particular lipid contents, it stains poorly or not at all with the usual lipid stains and so is not usually detected by standard lipoprotein electrophoretic patterns.
 
Lipoprotein Metabolism and Atherosclerosis
Coronary heart disease (CHD) is the most common cause of death. The key event in early atherosclerosis is damage to the endothelium, which is initially only functional. The endothelium becomes more permeable to lipoproteins and allows migration of cells to the underlying layer, the intima. The LDL particles penetrate the vascular wall and are deposited in the intima, where they may undergo oxidation. Oxidized LDL stimulates endothelial expression of adhesion molecules, such as vascular cell adhesion molecules and monocyte chemotactic proteins. These attract monocytes, which are the precursors of phagocytic macrophages to the arterial wall. Monocytes enter the wall and are transformed into macrophages under the influence of macrophage monocyte colony stimulating factor. Oxidized LDL facilitates this process by inhibiting the mobility of macrophages, thus immobilizing them in the subendothelial space. Oxidized LDL is ingested by macrophages through their scavenger receptors. Since the scavenger receptor are not regulated, the cells become overloaded with lipid to form foam cells. Conglomerates of foam cells form fatty streaks. Dying foam cells release lipid that forms pools within the arterial wall. Further damage occurs through smooth muscle and endothelial cells, which start to secrete cytokines, a range of small peptides regulating cell growth, as well as growth factors. These include platelet-derived growth factor, interleukin and tumor necrosis factor, which stimulate smooth muscle cells to proliferate and to migrate toward the luminal side of the arterial wall. At that time, smooth muscle cells start synthesizing extracellular matrix in particular collagen. The relocation of smooth muscle cells and accumulation of new matrix results in the formation of a cap that covers the lipid pool and consists of collagen rich fibrous tissue, smooth muscle cells, macrophages and T lymphocytes. Eventually, the final lesion is termed a mature atherosclerotic plaque. The presence of an atherosclerotic plaque in the artery causes increased rigidity in the vessels as well as a narrowing of the lumen. The consequences of atherosclerosis primarily result from impaired blood supply leading to ischemic heart disease (IHD). The effects of IHD range in severity from short-lasting chest pain of varying severity 205(angina pectoris) to more severe and more prolonged pains of myocardial infarction (MI). Angina results from ischemia, which is due to the imbalance between the heart's demands for oxygen and the poor blood supply. Such imbalance is accentuated by physical exertion, such as exercise.
Myocardial infarction, on the other hand, is due to the lack of blood supply, which leads to anoxic necrosis of part(s) of the myocardium. In MI, a nonfunctional, noncontractile fibrous tissue made of collagen fibrils replaces the normal contractile myocardium. The presence of this ectopic fibrous tissue can cause disruption of the normal heart's conductive system, which can lead to various, and sometimes life-threatening arrhythmias, such as ventricular fibrillation, probably responsible for the majority of deaths within the first few hours of the infarct.
A further consequence of atherosclerosis is thrombus formation. This event may be initiated by macrophages, residing at the edges of the plaque secreting metalloproteinases, which are enzymes that degrade the extracellular matrix. In addition, T cells produce g-interferon, which inhibits collagen synthesis in the smooth muscle cells. These processes weaken the plaque cap and may result in its breakage. The breakage exposes collagen and lipids to the blood stream. This leads to adherence and aggregation of platelets and initiates the formation of a blood clot or “thrombus”. Thrombus formation in the coronary arteries can produce thromboembolic effects on the cardiovascular system leading to blockage of various vessels, such as the cerebral arteries causing the cerebrovascular disease “stroke” as well as the myocardium causing CAD.
In addition to the above effects, which relate to atherosclerosis of the coronary arteries, atherosclerosis of other arteries can result in severe pathological symptoms. Atherosclerosis of peripheral arteries, e.g. femoral arteries in the limbs can lead to peripheral vascular disease. Peripheral vascular disease is characterized by ischemia to the lower limbs resulting in various symptoms, such as difficulty in walking long distances without pain, and in severe cases, gangrene from ischemic necrosis. It can be deduced from the above account that the effects of atherosclerosis are severe. However, the effects can be prevented to some extent by the reduction of blood cholesterol and TG levels. One way in which this can be achieved is by changes in the diet. In addition, atherosclerosis can be reduced by a reduction in smoking and hypertension or by an increase in exercise. One proposed mechanism by which changes in diet and exercise can reduce cholesterol and TG levels is by regulation of LPL activity. Decreased LPL activity can result in high blood lipids. Therefore, patients with decreased LPL activity are more prone to raised blood lipids and atherosclerosis.
 
MEASUREMENT OF LIPOPROTEINS
Measurement of lipoproteins may be performed by ultracentrifugation of the plasma. When plasma is subjected to very high centrifugal force, the lipoproteins can be separated in a gradient salt solution on the basis of their density. Since the density is directly related to protein content, lipoproteins can also be separated by electrophoresis.206
Electrophoresis is the separation of charged particles in an electrical field and is dependent on the amount and nature of the Apos within the lipoprotein. The electrophoretic positions of the lipoproteins are often used to describe them. Thus, HDL is also called α1-lipoprotein, VLDL is called pre-β-lipoprotein, and LDL is called β-lipoprotein. Chylomicrons do not migrate and are not given any designation. When one or more plasma lipid levels are extremely elevated or reduced, either of these methods may be used to determine which lipoproteins are abnormal. On the basis of these findings, abnormal lipoproteins are classified into patterns. Since severe disorders of lipoprotein metabolism are often inherited, the abnormal patterns are called phenotypes. There are five abnormal lipoprotein phenotypes (type I through type V), each characterized by the presence of an extremely high quantity of one or two lipoproteins. Persons with severe hyperlipoproteinemia often have skin and tissue infiltration of fat deposits and persons with type II and type III are predisposed to premature atherosclerosis owing to high levels of plasma cholesterol. Lipoprotein phenotyping is not performed as a screening test to evaluate risk of CAD.
Immunological methods are used to measure the quantity of specific Apos present in the plasma. Testing for Apo A I and Apo B 100, the principle Apos of HDL and LDL, respectively is often performed in persons with elevated lipids who have risk factors associated with CAD. Measurement of a form of LDL, called Lp (a) is performed on these persons as well as on those who have normal lipid levels, but a family history of CAD.
High density lipoprotein cholesterol is routinely measured along with total cholesterol and TG as a screening test for CAD. If the total cholesterol is 200 mg/dL or higher, the LDL cholesterol is measured. The measurement of HDL cholesterol is based upon the measurement of cholesterol (see entry on lipid tests) under conditions that inhibit the reaction with all lipoproteins except HDL. The measurement of LDL cholesterol involves precipitating the HDL, VLDL and CM using antibodies to Apo A and Apo E, followed by measurement of the LDL cholesterol in the supernatant. When the TG concentration is below 400 mg/dL, the LDL cholesterol is often estimated using the Friedewald formula [LDL cholesterol = total cholesterol minus (HDL cholesterol + TG/5)]. It should be noted that this formula will underestimate LDL cholesterol when TGs are above 400 mg/dL.
 
Indications
  • Evaluate known or suspected disorders associated with altered lipoprotein levels
  • Evaluate patients with serum cholesterol levels greater than 250 mg/dL, which indicate a high risk for CAD
  • Evaluate the response to treatment for high cholesterol and determine the need for drug therapy.
 
LIPOPROTEIN CLASSIFICATION
The differentiation of lipoproteins according to their mobility in zone electrophoresis is the basis of the Fredrickson classification (Tables 2 and 3) of 207hyperlipoproteinemia.
Table 2   Fredrickson classification
Hyperlipoproteinemia: Fredrickson type
Specimen appearance
Electrophoretic pattern
Type I
Clear with creamy top layer
Heavy chylomicron band
Type IIa
Clear
Heavy β band
Type IIb
Clear or faintly turbid
Heavy β and pre-β bands
Type III
Slightly to moderately turbid
Heavy β band
Type IV
Slightly to moderately turbid
Heavy pre-β band
Type V
Slightly to moderately turbid with creamy top layer
Intense chylomicron band and heavy β and pre-β bands
Table 3: Popular system of classification
208
This classification readily allows application of dietary measures or therapeutic treatment.
 
Primary Hyperlipoproteinemia
Familial hyperlipidemias are classified according to the Fredrickson classification, which is based on the pattern of lipoproteins on electrophoresis or ultracentrifugation.
Donald Fredrickson was born in Canon City, Colorado. His father was a county judge and the owner of Fredrickson Brown, an independent insurance agency. After high school, he commenced medical school at the University of Colorado, but completed his studies at the University of Michigan after being transferred there by the army. During a cycling trip in the Netherlands, he met his future wife, Priscilla Eekhof, and they married 2 years later. They would have two sons. Between 1949 and 1952, he worked as a resident and subsequently as a fellow in internal medicine at the Peter Bent Brigham Hospital (now part of Brigham and Women's Hospital) in Boston. Much of his published work from this period is in the field of endocrinology. Subsequently, he spent a year in the laboratory of Ivan Frantz, a cholesterol biochemist, at Massachusetts General Hospital.
It was later adopted by the World Health Organization (WHO). It does not directly account for HDL, and it does not distinguish among the different genes. It remains a popular system of classification (Tables 2 and 3).
  • Type I: Hyperlipoproteinemia or increased CM can be primary, resulting from an inherited deficiency of LPL, or secondary caused by uncontrolled diabetes, systemic lupus erythematosus and dys-γ-globulinemia. Total cholesterol is normal to moderately elevated, and TGs (mostly exogenous CM) are grossly elevated. If the condition is inherited, symptoms will appear in childhood.
  • Type IIa: Hyperlipoproteinemia can be primary, resulting from inherited characteristics, or secondary, caused by uncontrolled hypothyroidism, nephrotic syndrome and dys-γ-globulinemia. Total cholesterol is elevated, TGs are normal and LDL is elevated. If the condition is inherited, symptoms will appear in childhood.
  • Type IIb: Hyperlipoproteinemia can occur for the same reasons as in type IIa. Total cholesterol, TGs, and LDL are all elevated.
  • Type III: Hyperlipoproteinemia can be primary, resulting from inherited characteristics, or secondary, caused by hypothyroidism, uncontrolled diabetes, alcoholism and dys-γ-globulinemia. Total cholesterol and TGs are elevated, whereas LDL is normal.
  • Type IV: Hyperlipoproteinemia can be primary, resulting from inherited characteristics, or secondary, caused by poorly controlled diabetes, alcoholism, nephrotic syndrome, chronic renal failure and dys-γ-globulinemia. 209Total cholesterol is normal to moderately elevated, TGs are moderately to grossly elevated and LDL is normal.
  • Type V: Hyperlipoproteinemia can be primary, resulting from inherited characteristics, or secondary, caused by uncontrolled diabetes, alcoholism, nephrotic syndrome and dys-γ-globulinemia. Total cholesterol is normal to moderately elevated, TGs are grossly elevated and LDL is normal.
 
Secondary Hyperlipoproteinemia
Acquired hyperlipidemias (also called secondary dyslipoproteinemias) often mimic primary forms of hyperlipidemia and can have similar consequences. They may result in increased risk of premature atherosclerosis or, when associated with marked hypertriglyceridemia, may lead to pancreatitis and other complications of the chylomicronemia syndrome. The most common causes of acquired hyperlipidemia are:
  • Diabetes mellitus
  • Use of drugs, such as diuretics, β-blockers and estrogens.
Other conditions leading to acquired hyperlipidemia include:
  • Hypothyroidism
  • Renal failure
  • Nephrotic syndrome
  • Alcohol
  • Some rare endocrine disorders and metabolic disorders
Treatment of the underlying condition, when possible or discontinuation of the offending drugs usually leads to an improvement in hyperlipidemia. Specific lipid lowering therapy may be required in certain circumstances.
Another acquired cause of hyperlipidemia, although not always included in this category, is postprandial hyperlipidemia, a normal increase following ingestion of food.
 
Principle of the Test
  • The analysis is performed by electrophoresis on buffered (pH 8.5) agarose gels. The separated bands of lipoproteins are then stained with a lipid-specific Sudan black stain and the excess of stain is removed with an alcoholic solution
  • The resulting electrophoregrams can be evaluated visually for pattern abnormalities or by densitometry to obtain relative quantification of individual zones
  • The lipoproteins are circulating complexes constituted of lipids and proteins. The classification of lipoproteins is based on their properties that lend themselves to the techniques for their separation:
    • Density (ultracentrifugation)
    • Charge (zone electrophoresis)
    • Size (molecular filtration by polyacrylamide gel electrophoresis).
On agarose gels, the major lipoproteins separate into the following fractions (in the order of increasing mobility):210
  • Chylomicrons: These are very large molecules with high TG content, present as small particles in the serum and responsible for its opalescence. They normally remain at the application point
  • β-lipoproteins or LDL: They normally migrate in β-globulin position
  • Pre-β-lipoproteins or VLDL: They have molecular weight higher and density lower than LDL; they are more mobile than LDL and migrate in front of β-globulin position
  • α-lipoproteins or HDL: They are the fastest fraction of lipoproteins; they migrate in α-2-globulin position.
Electrophoresis is a simple and useful technique for the assessment of lipoprotein abnormalities and coronary disease risk factors in serum.
 
Methodology
 
Pretest
Positively identify the patient using at least two-unique identifiers before providing care, treatment, or services.
 
Patient Teaching
  • Inform the patient that this test can assist in evaluating cardiac health
  • Obtain a history of the patient's complaints including a list of known allergens, especially allergies or sensitivities to latex
  • Obtain a history of the patient's cardiovascular system, risk for heart disease, symptoms, and results of previously performed laboratory tests, and diagnostic and surgical procedures
  • Obtain a list of the patient's current medications including herbs, nutritional supplements
  • Review the procedure with the patient. Inform the patient that specimen collection takes approximately 5–10 minutes. Address concerns about pain and explain that there may be some discomfort during the venipuncture
  • Sensitivity to social and cultural issues, as well as concern for modesty, is important in providing psychological support before, during and after the procedure
  • Instruct the patient to follow his or her usual diet for 2 weeks before testing.
  • Instruct the patient to fast and to avoid excessive exercise for at least 12 hours before testing and to refrain from alcohol consumption for 24 hours before testing
  • There are no medication restrictions unless by medical direction.
 
Intratest
  • Ensure that the patient has complied with dietary and activity restrictions as well as other pretesting preparations; assure that food, fluids and activity have been restricted for at least 12 hours and that alcohol has been restricted for at least 24 hours prior to the procedure
  • If the patient has a history of allergic reaction to latex, avoid the use of equipment containing latex
  • Instruct the patient to cooperate fully and to follow directions211
  • Observe standard precautions and follow the general guidelines in patient preparation and specimen collection. Positively identify the patient and label the appropriate tubes with the corresponding patient demographics, date and time of collection. Perform a venipuncture
  • Remove the needle and apply direct pressure with dry gauze to stop bleeding. Observe/assess venipuncture site for bleeding or hematoma formation and secure gauze with adhesive bandage
  • Promptly transport the specimen to the laboratory for processing and analysis.
 
Post-test
  • A report of the results will be sent to the requesting health care provider who will discuss the results with the patient
  • Instruct the patient to resume usual diet, fluids and activity
  • Nutritional considerations: Overweight patients with high blood pressure should be encouraged to achieve a normal weight. Other changeable risk factors warranting patient education include strategies to safely decrease sodium intake, increase physical activity, decrease alcohol consumption, eliminate tobacco use and decrease cholesterol levels
  • Social and cultural considerations: The medical, social and emotional consequences of excess body weight are significant. Special attention should be given to instructing the child and caregiver regarding health risks and weight control education
  • Recognize anxiety related to test results, and be supportive of fear of shortened life expectancy. Discuss the implications of abnormal test results on the patient's lifestyle. Provide teaching and information regarding clinical implications of the test results as appropriate. Educate the patient regarding access to counseling services.
 
Collection and Handling of Specimens
The patient should be fasting for a 12–14-hour period since CM normally appear in the blood 2–10 hours after a meal. Sample should be collected into a vacutainer tube containing 1.5 mg Na2EDTA per mL blood. The lipoproteins are generally instable; to protect them, the serum or plasma should be separated from the red blood cells within 2 hours after collection.
The β- and α-lipoproteins remain relatively constant over at least 28 days at 4°C; however, there is a decrease in the mobility of the pre-β-lipoproteins, and the change is most rapid in the first 5 days. Freezing of the plasma irreversibly alters the lipoprotein pattern and, therefore, it must be stored at 4°C.
 
Serum Storage
For best results, fresh serum should be used. Storage at 2–8°C for no more than 5 days yields satisfactory results. Prolonged storage increases the migration rate of the pre-β fraction. Sample should not freeze.212
 
Reagents and Materials
 
Agarose Gel
Agarose gels are ready to use. Each gel contains agarose, 0.8 g/dL; buffer pH 8.5 ± 0.1; additives, nonhazardous at concentrations used, necessary for optimum performance.
Storage, stability and signs of deterioration: Store the gels horizontally in the original protective packaging at room temperature (15–30°C) or refrigerated (2–8°C) (the arrow on the front of the kit box must be pointing upwards). The gel should not be kept frozen; obvious temperature fluctuations during storage (e.g. do not store close to a window or to a heat source) should be avoided. The gels are stable until the expiration date indicated on the kit package or the gel package labels.
Agarose gel should be discarded under certain conditions:
  • Crystals or precipitate form on the gel surface or the gel texture becomes very soft (all these result from freezing the gel)
  • Bacterial or mold growth is indicated
  • Abnormal quantity of liquid is present in the gel box (as a result of buffer exudation from the gel due to improper storage conditions).
 
Sudan Black Stain
Working Sudan black stain solution should be prepared at least 30 minutes before use. Exact volumes of the individual components in the following order should be added under gentle stirring:
  1. Pure ethanol (96%), 120 mL; Sudan black stain, stock solution (6.6 g/dL in dimethylformamide), 1.45 mL. Wait until the Sudan black is completely dissolved and then add distilled or deionized water, 100 mL. Stir for a minimum 30 minutes, or
  2. Pure isopropanol (100%), 100 mL; Sudan black stain, stock solution (6.6 g/dL in dimethylformamide), 1.45 mL. Wait until the Sudan black is completely dissolved and then add distilled or deionized water, 120 mL. Stir for a minimum 30 minutes.
These solutions should be kept away from any source of heat and should be discarded after each staining procedure. The use of another alcohol or denatured ethanol may lead to atypical results. If lower concentrations than indicated of pure alcohol are used, the volumes should be adjusted.
 
Destaining Solutions
The destaining solutions (v/v) should be prepared at least 15 minutes before use:
  1. 45% pure ethanol (96 %) and 55% distilled or deionized water
  2. 30% pure isopropanol (100%) and 70% distilled or deionized water.
The use of another alcohol or denatured ethanol may lead to atypical results. If lower concentrations than indicated of pure alcohol are used, adjust accordingly the volumes of alcohol and water.213
 
Wash Solutions
  1. 75% pure ethanol (96%) and 25% distilled or deionized water, or
  2. 70% pure isopropanol (100%) and 30% distilled or deionized water.
Denatured ethanol may also be used. If lower concentrations than indicated of alcohol are used, adjust accordingly the volumes of alcohol and water. Wash solution can be stored at room temperature tightly capped to prevent evaporation. It is stable for 3 months at room temperature. Discard destaining solution if it changes its appearance, e.g. becomes cloudy due to microbial contamination.
 
Quality Control
It is advised to include an assayed serum with normal TGs and cholesterol levels into each run of samples.
 
Values
Densitometry of stained electrophoregrams at 570 nm or with yellow filter yields relative concentrations (percentages) of each fraction.
  • β-lipoproteins (LDL): 42.3–69.5%
  • Pre-β-lipoproteins (VLDL): 2.0–31.2%
  • α-lipoproteins (HDL): 15.1–39.9%.
 
Reproducibility
In all reproducibility studies, the electrophoregram is to be scanned and relative percent concentration of each lipoprotein fraction is to be recorded. Means, standard deviation (SD), and coefficient of variation (CV) should be calculated with respect to the parameters examined.
 
Gel-to-gel Reproducibility
Different samples should be analyzed; each sample to be applied in one track in each of the gels from the same lot. The mean, SD and CV percentages are to be calculated for each sample and each lipoprotein fraction.
 
Interferences and Limitation
Heparin therapy causes activation of LPL, which increases the relative migration rates of the fractions, especially the β-lipoprotein.
  • Failure to follow usual diet for 2 weeks before the test can yield results that do not accurately reflect the patient's cholesterol values
  • Ingestion of alcohol 24 hours before the test, ingestion of food 12 hours before the test and excessive exercise 12 hours before the test can alter results
  • Numerous drugs can alter results
  • Failure to follow dietary restrictions before the procedure may cause the procedure to be canceled or repeated
  • Frozen samples should be avoided214
  • Storage causes slow down of pre-β-lipoprotein mobility, and, therefore, under evaluation of this fraction. This becomes the most apparent with samples low in VLDL (<10%).
  • In some sera, a more or less diffuse trail may appear in front of α-lipoprotein fraction (HDL). This zone corresponds to partially delipidated HDL, and it has to be included in the HDL percentage.
 
Interpretation of Lipoprotein Patterns
The lipoprotein pattern of a clinical sample must be interpreted visually by comparing it with a control or a normal serum pattern. Densitometry provides accurate relative percentage of individual lipoprotein fractions. Such quantitation may be useful for the follow-up of the patient by monitoring the fractions' changes. The relative (%) values obtained by densitometry, however, cannot be used to type hyperlipidemia. Qualitative (presence of abnormal or absence of normal fractions) or semiquantitative (relative increase or decrease of fractions) abnormalities necessitate further lipoprotein analyzes.
 
Reference Values
Total cholesterol guidelines are:
  • Desirable: <200 mg/dL
  • Borderline high: 200–239 mg/dL
  • High: >240 mg/dL
High density lipoprotein guidelines reflect the fact that this lipoprotein is inversely related to the risk for CAD.
Low density lipoprotein cholesterol guidelines are:
  • Low: <40 mg/dL
  • Desirable: <130 mg/dL
  • Optimal: <100 mg/dL
  • Near optimal: 100–129 mg/dL
  • Borderline high: 130–159 mg/dL
  • High: 160–189 mg/dL
  • Very high: >190 mg/dL
Identified factors that make the risk of heart disease higher can help the patient to lower their cholesterol as much as possible:
  • Cigarette smoking
  • High blood pressure with a measurement of greater than 140/90 mmHg. In addition, it is considered a risk factor if the patients are on blood pressure lowering medications even if they have achieved a normal blood pressure
  • Age over 45 years in men and over 55 years in women. Estrogen, a sex hormone in women protects against heart disease. The levels of estrogen are lower after a woman goes through menopause, roughly after the age of 55
  • Low HDL cholesterol (< 35 mg/dL). Note that HDL greater than 60 mg/dL is a negative risk factor for CAD215
  • Family history of premature heart disease: Premature heart disease is defined as heart disease seen before age 55 in a male relative or before age 65 in a female relative
  • Diabetes mellitus.
The α-lipoprotein (HDL) band is the fastest moving fraction and is located closest to the anode. The β-lipoprotein (LDL) band is usually the most prominent fraction and is near the origin, migrating only slightly anodic to the point of application. The pre-β-lipoprotein (VLDL) band migrates between α and β lipoprotein. The mobility of pre-β-lipoproteins varies with the degree of resolution obtained, the type of pre-β present and the percent of β present. Sometimes pre-β will be seen as a smear just ahead of the fraction. Other times it may be split into two or more fractions or may be lacking altogether. The integrity of the pre-β fraction decreases with sample age. Chylomicrons, when present, stay at the point of application. In samples with very high levels of CM, there will appear to be a smear of material extending anodically from the point of applications to the β band.
Normal pattern: A normal fasting serum can be defined as a clear serum with negligible CM and normal cholesterol and TG levels. On electrophoresis, the β-lipoprotein appears as the major fraction, with the pre-β-lipoprotein faint or absent and the α band definite but less intense than the β band (Fig. 2).
Abnormal patterns: A patient must have an elevated cholesterol or TG to have hyperlipoproteinemia (Fig. 3). The elevation must be determined to be primary or secondary to metabolic disorders, such as hypothyroidism, obstructive jaundice, nephrotic syndrome, dysproteinemia or poorly controlled insulinopenic diabetes mellitus.
Fig. 2: Normal fasting serum lipoprotein electrophoresis fractions
216
Fig. 3: Abnormal patterns of hyperlipoproteinemia
Primary lipidemia arises from genetically determined factors or environmental factors of unknown mechanism, such as diet, alcohol intake and drugs, especially estrogen or steroid hormones. Also considered primary are those lipoproteinemias associated with ketosis resistant diabetes, pancreatitis and obesity. Diabetes mellitus and pancreatitis can be confusing, for it is often difficult to tell whether the hyperlipoproteinemia or the disease is the causative factor.
 
Primary Hyperlipoproteinemia
 
Type I: Hyperchylomicronemia
  • Chylomicrons present, pre-β normal or only slightly elevated, α and β decreased, often markedly so standing plasma with marked creamy layer
  • The confirmation is done by the measurement of postheparin lipolytic activity (PHLA) and demonstration of severe intolerance to exogenous fat
  • The condition is rare and always familial. There has been no correlation to vascular disease. It is thought to be due to a genetic deficiency of LPL (Fig. 4).217
Fig. 4: Type I: Hyperchylomicronemia
Fig. 5: Type IIa: Hyperlipoproteinemia
 
Type II: Hyperlipoproteinemia
Hyperlipoproteinemia is the increased total cholesterol due to an increased β-lipoprotein cholesterol. The α-cholesterol is usually normal or low.
  • Type IIa: Normal pre-β, normal TGs, plasma clear (Fig. 5)
  • Type IIb: Increased pre-β and TGs, plasma clear to slightly turbid with no creamy layer (Fig. 6).
This is one of the most common familial forms of hyperlipoproteinemia. The secondary causes are myxedema, myelomas, macroglobulinemia, nephrosis, liver disease, and excesses in dietary cholesterol, and saturated fats.
 
Type III: “Broad B” Abnormal Lipoprotein
It is characterized by presence of TG-burdened lipoprotein of abnormal composition and density. Cholesterol and TG are elevated. The abnormal material has broad β-electrophoretic mobility but separates with VLDL in 218the ultracentrifuge.
Fig. 6: Type IIb hyperlipoproteinemia
Fig. 7: Type III hyperlipoproteinemia
Plasma is turbid to cloudy. The abnormal lipoprotein is also known as “floating B”. The condition is rare. The confirmation is done by ultracentrifuge study to demonstrate the abnormal lipoprotein (Fig. 7).
 
Type IV: Carbohydrate-induced and Endogenous Hypertriglyceridemia
  • Increased pre-β, increased TGs, normal or slightly increased total cholesterol, α- and β-lipoprotein usually normal (an increased pre-β with normal TG level is seen (Fig. 8) with the normal variant “sinking pre-β”. Such samples do not belong to Type IV)
  • The secondary causes include nephrotic syndrome, diabetes mellitus, pancreatitis, glycogen storage disease and other acute metabolism changes where mobilization of free fatty acids is increased
  • Endogenous TGs are very sensitive to alcohol intake, emotional stress, diet and changes in weight. Little effect is seen with exogenous TG intake. Type IV has an abnormal glucose tolerance. Probably, it is the most common type of hyperlipoproteinemia reflecting an imbalance in synthesis and clearance of endogenous TGs.219
Fig. 8: Type IV hyperlipoproteinemia
Fig. 9: Type V hyperlipoproteinemia
 
Type V: Mixed Triglyceridemia
  • Increased exogenous and endogenous TGs, cholesterol increased, CM present, pre-β increased, β normal to slightly increased
  • The secondary causes include nephrosis, myxedema, diabetic acidosis, alcoholism, pancreatitis, glycogen storage disease, and other acute metabolic processes (Fig. 9).
 
The α-lipoprotein in Disease
Marked increase in α-lipoproteins is seen in obstructive liver disease and cirrhosis. Marked decreases are seen in parenchymal liver disease. Tangier's disease is a rare genetic disorder characterized by the total absence of normal α-lipoproteins. Heterozygotes exhibit decreased levels of α-lipoproteins. It should be noted that hyperestrogenemia (pregnancy and oral contraceptive use) may cause moderate elevations in α-lipoproteins.
 
Decreases in the β-lipoprotein
Abetalipoproteinemia or Bassen Kornzweig syndrome is a rare autosomal recessive disorder that interferes with the normal absorption of fat and fat-soluble vitamins from food. It is caused by a mutation in microsomal 220triglyceride transfer protein (MTTP) resulting in deficiencies in the Apos B 48 and B 100 that are used in the synthesis and exportation of CM and VLDL, respectively. It is not to be confused with familial dys-β-lipoproteinemia. Abetalipoproteinemia affects the absorption of dietary fats, cholesterol and certain vitamins. People affected by this disorder are not able to make certain lipoproteins that are molecules that consist of proteins combined with cholesterol and particular fats called TGs. This leads to a multiple vitamin deficiency affecting the fat-soluble vitamin A, vitamin D, vitamin E and vitamin K. However, many of the observed effects are due to vitamin E deficiency in particular.
The signs and symptoms of abetalipoproteinemia appear in the first few months of life (because pancreatic lipase is not active in this period). They can include failure to gain weight and grow at the expected rate (failure to thrive), diarrhea, abnormal star-shaped red blood cells (acanthocytosis) (Fig. 10) and fatty, foul-smelling stools (steatorrhea). The stool may contain large chunks of fat and/or blood. Other features of this disorder may develop later in childhood and often impair the function of the nervous system. They can include poor muscle coordination, difficulty with balance and movement (ataxia), and progressive degeneration of the light sensitive layer (retina) at the back of the eye that can progress to near blindness (due to the deficiency of vitamin A, retinol). Adults in their thirties or forties may have increasing difficulty with balance and walking. Many of the signs and symptoms of abetalipoproteinemia result from a severe vitamin deficiency, especially vitamin E deficiency, which typically results in eye problems with degeneration of the spinocerebellar and dorsal columns tracts.
 
Symptoms
Often symptoms will arise that indicate the body is not absorbing or making the lipoproteins it needs. These symptoms usually appear en masse, meaning that they happen all together, all the time. These symptoms come as follows:
  • Failure to grow in infancy
  • Fatty, pale stools
  • Frothy stools
Fig. 10: Acanthocytosis in a patient with abetalipoproteinemia
221
222
Fig. 11: Autosomal recessive pattern of inheritance in abetalipoproteinemia
  • Foul-smelling stools
  • Protruding abdomen
  • Mental retardation /developmental delay
  • Dyspraxia, evident by age 10
  • Muscle weakness
  • Slurred speech
  • Scoliosis (curvature of the spine)
  • Progressive decreased vision
  • Balance and coordination problems
  • Retinitis pigmentosa.
 
Genetics
Mutations in the MTTP gene have been associated with this condition (Apo B deficiency, a related condition, is associated with deficiencies of Apo B). The MTTP gene provides instructions for making a protein called MTTP, which is essential for creating β-lipoproteins. These lipoproteins are necessary for the absorption of fats, cholesterol and fat-soluble vitamins from the diet and for the efficient transport of these substances in the blood stream. Most of the mutations in this gene lead to the production of an abnormally short MTTP, which prevents the normal creation of β-lipoproteins in the body. MTTP-associated mutations are inherited in an autosomal recessive pattern, which means both copies of the gene must be faulty to produce the disease (Fig. 11).
 
Treatment
Treatment normally consists of rigorous dieting involving mass amounts of vitamin E. Vitamin E helps the body restore and produce lipoproteins, which people with abetalipoprotenemia usually lack. Vitamin E also helps keep skin and eyes healthy. Studies show that many affected males will have vision problems later on in life. Dyspraxia and muscle weakness are usually treated with physiotherapy or occupational therapy. Dietary restriction of TGs has also been useful.

Alkaline Phosphatase ElectrophoresisCHAPTER 8

 
INTRODUCTION
Alkaline phosphatase (ALP) [Enzyme Commission (EC) 3.1.3.1 )] is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, ALPs are most effective in an alkaline environment. It is sometimes used synonymously as basic phosphatase. It is a metalloglycoprotein with a monophophoesterase activity, and it catalyzes the hydrolysis of monophosphoric esters, which is activated by magnesium ions in alkaline medium.
 
PHYSIOLOGY
Alkaline phosphatase is an enzyme found in the liver in Kupffer's cells lining the biliary tract, and in bones, intestines, and placenta. Additional sources of ALP include the proximal tubules of the kidneys, pulmonary alveolar cells, germ cells, vascular bed, lactating mammary glands, and granulocytes of circulating blood.
In humans, ALPs are a group of enzymes found primarily in the liver (isoenzyme ALP) and bone (isoenzyme ALP). There are also small amounts produced by cells lining the intestines (isoenzyme ALP), the placenta, and the kidney (in the proximal convoluted tubules). What is measured in the blood is the total amount of ALP released from these tissues into the blood. As the name implies, this enzyme works best at an alkaline medium (pH of 9.1 ± 0.05), and, thus, the enzyme itself is inactive in the blood. Alkaline phosphatases act by splitting off phosphorus (an acidic mineral) creating an alkaline pH.224
Its elevation is evident in a variety of hepatic, nonhepatic conditions as well as in normal adolescent growth. The enzymes of ALP are coded by three structural genes: two genes code respectively for the placental and intestinal isoenzymes. The third gene called “nonspecific tissue gene” is expressed in a variety of tissues, such as bone, the liver and kidney.
The primary importance of measuring ALP is to check the possibility of bone or liver disease. Since the mucosal cells that line the bile system of the liver are the source of ALP, the free flow of bile through the liver and down into the biliary tract and gallbladder are responsible for maintaining the proper level of this enzyme in the blood. When the liver, bile ducts or gallbladder system are not functioning properly or are blocked, this enzyme is not excreted through the bile and ALP is released into the blood stream. Thus the serum ALP is a measure of the integrity of the hepatobiliary system and the flow of bile into the small intestine.
In addition to liver, bile duct, or gallbladder dysfunction, an elevated serum ALP can be due to rapid growth of bone since it is produced by bone forming cells called osteoblasts. One would expect that growing children have higher levels than full-grown adults. The relationship of alkalinity to bone development warrants further discussion because it plays a major role in the prevention and reversal of osteoporosis. Just as calcium builds up around faucets, so is calcium laid down into bone. The reason the calcium deposits on faucet is because the water is alkaline and calcium comes out of solution and crystallizes in an alkaline environment. The reverse is also true. “Lime away”, vinegar, or any other acidic solution dissolves the calcium deposits because they are acidic. It makes sense that osteoblasts by creating a local environment of alkalinity via ALP helps build bone. It also implies that in order to slow bone loss, one cannot be in an acidic state.
Because acid-alkaline is influenced by many other glands, the implications of serum ALP levels must consider more than just bone and liver function. Associated organs/glands include adrenals, uterus, prostate, and intestine.
The optimal range for ALP depends on the age. A growing adolescent will have a much higher ALP than a full-grown adult.
 
INDICATIONS
The primary reason for analyzing ALP isoenzymes is to determine whether an increased level of ALP is due to bone or liver enzyme. If the sample exhibits any other liver enzyme changes [often γ-glutamyl transpeptidase (GT) elevation] it is usual but not inevitable, that any excess ALP is derived from the liver. Therefore, if there is an isolated increase in ALP and no other information exists that suggests where it is derived from, and it is felt necessary to investigate the high levels, the appropriate investigation is ALP isoenzymes.
Evaluate signs and symptoms of various disorders associated with elevated ALP levels, such as biliary obstruction, hepatobiliary disease, and bone disease, including malignant processes.225
Differentiate obstructive hepatobiliary tract disorders from hepatocellular disease. Greater elevations of ALP are seen in the former.
Determine effects of renal disease on bone metabolism.
Determine bone growth or destruction in children with abnormal growth patterns.
 
DIAGNOSTIC USE
If it is unclear why ALP is elevated, isoenzyme studies using electrophoresis can confirm the source of the ALP. Heat stability also distinguishes bone and liver isoenzymes. Placental ALP is elevated in seminomas and active form of rickets. High ALP levels can show that the bile ducts are blocked. Levels are significantly higher in children and pregnant women. Also, elevated ALP indicates that there could be active bone formation occurring as ALP is a byproduct of osteoblast activity (such as the case in Paget's disease of bone). Levels are also elevated in people with untreated celiac disease. Lowered levels of ALP are less common than elevated levels.
 
Elevated Levels
It is related to release of ALP from damaged bone, biliary tract, and liver cells. Congestion or obstruction of the biliary tract, which may occur within the liver, the ducts leading from the liver to the gallbladder, or the duct leading from the gallbladder through the pancreas that empty into the duodenum (small intestine). Any of these organs (liver, gallbladder, pancreas, or duodenum) may be involved.
  • Liver congestion/cholestasis
  • Oral contraceptives
  • Obstructive pancreatitis
  • Hepatitis/mononucleosis/cytomegalovirus (CMV)
  • Congestive heart failure
  • Parasites
  • Malignancy involving liver
  • Osteoblastic/bone conditions
  • Paget's disease
  • Herpes zoster (Shingles)
  • Hyperthyroidism [Overactivity of the parathyroid glands (primary hyperparathyroidism, secondary hyperparathyroidism from kidney disease, osteomalacia, malabsorption]
  • Rickets: Vitamin D deficiency
  • Healing fractures, rapid bone growth such as after a fracture, bone cancers like osteogenic sarcoma, osteomalacia, and Paget's disease.
  • Osteoporosis treatment
  • Adrenal cortical hyperfunction
  • Non-bone/non-liver conditions
  • As a normal part of late pregnancy since the placenta produces ALP (placenta ~2 x normal)
  • Amyloidosis226
  • Granulation tissue
  • Gastrointestinal inflammation [inflammatory bowel disease (ulcerative colitis, Crohn's disease; ulcers)]
  • Systemic infections (sepsis)
  • Sarcoidosis
  • Rheumatoid arthritis
  • Certain cancers, such as Hodgkin's lymphoma, gynecologic malignancies
  • Acute tissue damage in the heart or lungs (myocardial or pulmonary infarctions).
 
Lowered Levels
The following conditions or diseases may lead to reduced levels of ALP:
  • Hypophosphatasia, an autosomal recessive disease
  • Postmenopausal women receiving estrogen therapy because of osteoporosis
  • Men with recent heart surgery
  • Children with achondroplasia and cretinism
  • Children after a severe episode of enteritis
  • Pernicious anemia
  • Aplastic anemia
  • Chronic myelogenous leukemia
  • Wilson's disease
  • Celiac disease
  • Human immunodeficiency virus (HIV)-1 infection
  • Folic acid deficiency
  • Hypervitaminosis D
  • Hypothyroidism (characteristic in infantile and juvenile cases)
  • Nutritional deficiency of zinc or magnesium
  • Scurvy (related to vitamin C deficiency)
  • Whipple's disease
  • Zollinger-Ellison syndrome.
  • In addition, the following drugs have been demonstrated to reduce ALP:
    • Oral contraceptives
    • Leukocyte alkaline phosphatase: Leukocyte alkaline phosphatase (LAP) is found within white blood cells. White blood cell levels of LAP can help in the diagnosis of certain conditions. Higher levels are seen in polycythemia vera (PV), essential thrombocytosis (ET), primary myelofibrosis (PM), and the leukemoid reaction. Lower levels are found in chronic myelogenous leukemia (CML), paroxysmal nocturnal hemoglobinuria (PNH) and acute myelogenous leukemia (AML).
 
Methodology
Routine measurement of ALP gives total serum activity (all isoforms) without specificity as to source. Isoform measurement is most commonly applied to 227distinguish ALP isoforms in cases with increased total serum ALP activity of uncertain cause (to identify whether total ALP is increased due to liver disease or endogenous corticosteroids). Differentiation of isoforms can be accomplished by electrophoretic (affinity agarose electrophoresis, cellulose acetate electrophoresis or isoelectric focusing on agarose or differential inhibition).
The individual ALP isoenzymes can normally be separated by electrophoresis according to the charge differences. However, the electrophoretic mobilities of the liver and bone isoenzymes are induced only by glycosylation or post-translational modifications and therefore are quite similar. They must be separated with a special treatment of the sample (Figs 1A and B). Quantitation of the ALP isoenzymes helps to identify the tissues responsible for the elevation.
Figs 1A and B: Alkaline phosphatase (ALP) isoenzyme electrophoresis separated with a special treatment
228
 
Pretest
  • Positively identify the patient using at least two unique identifiers before providing care, treatment, or services
  • Obtain a history of the patient's complaints, including a list of known allergens, especially allergies or sensitivities to latex
  • Obtain a history of the patient's hepatobiliary and musculoskeletal systems, symptoms, and results of previously performed laboratory tests and diagnostic and surgical procedures
  • Review the procedure with the patient. Inform the patient that specimen collection takes approximately 5–10 minutes. Address concerns about pain and explain that there may be some discomfort during the venipuncture
  • There are no food, fluid, or medication restrictions unless by medical direction.
 
Intratest
  • If the patient has a history of allergic reaction to latex, avoid the use of equipment containing latex
  • Instruct the patient to cooperate fully and to follow directions. Direct the patient to breathe normally and to avoid unnecessary movement
  • Observe standard precautions, and follow the general guidelines in Patient Preparation and Specimen Collection. Positively identify the patient, and label the appropriate tubes with the corresponding patient demographics, date, and time of collection. Perform a venipuncture
  • Remove the needle and apply direct pressure with dry gauze to stop bleeding. Observe/assess venipuncture site for bleeding and hematoma formation and secure gauze with adhesive bandage
  • Promptly transport the specimen to the laboratory for processing and analysis.
 
Post-test
  • A written report of the results will be sent to the requesting doctor who will discuss the results with the patient
  • Increased ALP levels may be associated with liver disease. Dietary recommendations may be indicated and vary depending on the severity of the condition. A low-protein diet may be in order if the patient's liver has lost the ability to process the end products of protein metabolism
  • Depending on the results of this procedure, additional testing may be performed to evaluate or monitor progression of the disease process and determine the need for a change in therapy
  • Evaluate test results in relation to the patient's symptoms and other tests performed.
 
Why the Test is Performed?
When total ALP test result is high, the doctor may order the ALP isoenzyme test. This test will help determine what part of the body is causing higher ALP levels.229
This test may be used to diagnose:
  • Bone disease
  • Cause of pain in the abdomen
  • Liver, gallbladder, or bile duct disease
  • Parathyroid gland disease
  • Vitamin D deficiency.
It may also be done to check liver function and effects of the medicines on the liver.
 
How to Prepare for the Test?
Patient should be fasting for 10–12 hours before the test, unless otherwise instructed by the doctor. Many drugs affect the level of ALP in the blood. Any medicine should not be stopped without instructing the doctor.
  • Allopurinol
  • Antibiotics
  • Anti-inflammatory medicines
  • Birth control pills
  • Certain arthritis drugs
  • Certain diabetes medicines
  • Chlorpromazine
  • Cortisone
  • Male hormones
  • Methyldopa
  • Narcotic pain medicines
  • Propranolol
  • Tranquilizers
  • Tricyclic antidepressants.
 
Principle of the Test
The human ALP enzyme is part of a group of enzymes (phosphoric monoester hydrolases) that catalyze the hydrolysis and transfer of a phosphate group at alkaline pH.
The ALP isoenzyme can normally be separated by electrophoresis according to the charge differences in a buffered agarose gel. After electrophoresis, the isoenzymes in the gel are detected by the following specific colorimetric chemical reaction. The indigo blue dye is formed at the site of each isoenzyme band. This pattern may be visually interpreted or scanned with a densitometer (Table 1 and Fig. 3).
Table 1   Normal pattern fractions of alkaline phosphatase (ALP) isoenzyme
Fraction
Isoenzyme
%
α1
Biliary
0
α2
Liver
52
β1
Bone
48
β2
Intestine
0
230
Fig. 2: Normal alkaline phosphatase (ALP) isoenzyme pattern
 
SAMPLE COLLECTION AND STORAGE
Fresh serum (1 mL) collected in a plain tube. Plasma (1 mL) collected in heparinized tube is also acceptable. Samples must be collected according to established procedures used in clinical laboratory testing. Allow serum specimen to clot completely at room temperature. Separate serum or plasma from cells as soon as possible or within 2 hours of collection and transfer 2 mL serum or plasma to a standard transport tube and store at 2–8°C as soon as possible after collection, and for up to 1 week, or keep it frozen.
 
Unacceptable Samples
  • Specimens collected in ethylenediamine tetraacetic acid (EDTA), sodium fluoride, or potassium oxalate.
  • Grossly hemolysed or lipemic specimens.
 
REAGENTS AND MATERIALS
 
Agarose Gel
Agarose gels are ready to use. Each gel contains agarose (1 g/dL), alkaline buffer pH 9.1 ± 0.05 and additives; nonhazardous at concentrations used; necessary for optimum performance.
 
Storage, Stability and Signs of Deterioration
Store the gels horizontally in the original protective packaging at room temperature (15–30°C) or refrigerated (2–8°C). (The arrow on the front of the kit box must be pointing upwards). The gel should not be kept frozen; obvious temperature fluctuations during storage (e.g. do not store close to a window or to a heat source) should be avoided. The gels are stable until the expiration date indicated on the kit package or the gel package labels.231
Agarose gel should be discarded under certain conditions:
  • Crystals or precipitate form on the gel surface or the gel texture becomes very soft (all these result from freezing the gel)
  • Bacterial or mold growth is indicated
  • Abnormal quantity of liquid is present in the gel box (as a result of buffer exudation from the gel due to improper storage conditions).
 
Lectin
It is used for the treatment of the migrating serum samples during electrophoretic separations. The lectin vial contains wheat germ agglutinin (WGA), which is available commercially in a stabilized lyophilized form. The lectin vial should be reconstituted with exactly 0.5 mL of saline and mixed gently and let it stand at room temperature for 5 minutes. Then mix gently to obtain clear solution of lectin that is ready to use.
 
Storage, Stability and Signs of Deterioration
Store the lyophilized lectin refrigerated (2–8°C). It is stable until the expiration date indicated on the kit package or lectin vial label. After reconstitution, the lectin solution must be stored at –20°C.
 
Substrate
It is used for the preparation of the visualization solution. It contains 5-bromo-4-chloro 3-indolyl phosphate (BCIP) in aminomethyl propanol (AMP) buffer pH 10.0, and additives,
It is nonhazardous at concentrations used, necessary for optimum performance. The visualization solution must be prepared away from light and just before use.
 
Storage, Stability and Signs of Deterioration
Store substrate at room temperature or refrigerated. It is stable until the expiration date indicated on the kit package or substrate vial label. The substrate may show mild crystallization without any adverse effects on its performance.
 
Chromogen
It is used for the preparation of the developing solution. It contains nitro-blue tetrazolium (NBT) in water.
 
Storage, Stability and Signs of Deterioration
Store chromogen at room temperature or refrigerated. It is stable until the expiration date indicated on the kit package or chromogen vial label.
 
Destaining Solution
It is used for washing the gel after enzymatic visualization and drying, and for rinsing of the staining compartment. The destaining solution contains citric acid, 50 mg/dL.232
 
Storage, Stability, and Signs of Deterioration
Store the stock destaining solution at room temperature or refrigerated. It is stable until the expiration date indicated on the kit package or destaining solution vial labels.
Working destaining solution is stable for 1 week at room temperature in a closed bottle.
 
Quality Controls
It is recommended that fresh normal sera or commercially available quality control sera be included in each electrophoretic procedure.
 
Values
The enzymatic activity of each fraction can be calculated from the densitometric percent values for each fraction and the total ALP activity.
 
NORMAL RESULTS
In normal individuals, ALP activity as well as isoenzyme distribution is age, sex, genetic and pregnancy dependent. Bone isoenzyme is elevated in children and in adults over age of 50 years (Table 2). Individuals who are B or O blood type and are secretor positive may have elevated intestinal isoenzymes, particularly after a fatty meal. Placental isoenzyme is elevated during and shortly after termination of pregnancy.
Note: It is recommended that each laboratory establish its own normal range.
 
INTERFERING FACTORS
Drugs that may increase ALP levels by causing cholestasis include anabolic steroids, erythromycin, ethionamide, gold salts, imipramine, interleukin 2, isocarboxazid, nitrofurans, oral contraceptives, phenothiazines, sulfonamides, and tolbutamide.
Drugs that may increase ALP levels by causing hepatocellular damage include acetaminophen (toxic), amiodarone, anticonvulsants, arsenicals, asparaginase, bromocriptine, captopril, cephalosporins, chloramphenicol, enflurane, ethionamide, foscarnet, gentamicin, indomethacin, lincomycin, methyldopa, naproxen, nitrofurans, probenecid, procainamide, progesterone, ranitidine, tobramycin, tolcapone, and verapamil.
Drugs that may cause an overall decrease in ALP levels include alendronate, azathioprine, calcitriol, clofibrate, estrogens with estrogen replacement therapy, and ursodiol.
Hemolyzed specimens may cause falsely elevated results.
Elevations of ALP may occur if the patient is nonfasting, usually 2–4 hours after a fatty meal, and especially if the patient is a Lewis positive secretor of blood group B or O. 233
Table 2   Alkaline phosphatase (ALP) isoenzyme reference range
Age/gender
Bone fraction
Liver fraction
1–5 yr
Male
39–308 units/L
< 8–101 units/L
Female
56–300 units/L
< 8–53 units/L
6–7 yr
Male
50–319 units/L
< 8–76 units/L
Female
56–300 units/L
< 8–53 units/L
8 yr
Male
50–258 units/L
< 8–62 units/L
Female
78–353 units/L
< 8–62 units/L
9–12 yr
Male
78–339 units/L
< 8–81 units/L
Female
78–353 units/L
< 8–62 units/L
13 yr
Male
78–389 units/L
< 8–48 units/L
Female
28–252 units/L
< 8–50 units/L
14 yr
Male
78–389 units/L
< 8–48 units/L
Female
31–190 units/L
< 8–48 units/L
15 yr
Male
48–311 units/L
< 8–39 units/L
Female
20–115 units/L
< 8–53 units/L
16 yr
Male
48–311 units/L
< 8–39 units/L
Female
14–87 units/L
< 8–50 units/L
17 yr
Male
34–190 units/L
< 8–39 units/L
Female
17–84 units/L
< 8–53 units/L
18 yr
Male
34–146 units/L
< 8–39 units/L
Female
17–84 units/L
< 8–53 units/L
19 yr
Male
25–123 units/L
< 8–39 units/L
Female
17–84 units/L
< 8–53 units/L
20 yr
Male
25–73 units/L
< 8–48 units/L
Female
17–56 units/L
< 8–50 units/L
Adult
Male
11–73 units/L
0–93 units/L
Female
11–73 units/L
0–93 units/L
Method: Inhibition/electrophoresis for fractionation.
Values may be slightly elevated in older adults.
234
 
INTERPRETATION OF RESULTS
 
Liver Isoenzyme
The major liver isoenzyme is seen in α2 position. The α2 liver ALP increases in the blood early in liver disease before most other liver function tests show abnormalities. An extensive group of conditions which led to the increased α2 liver ALP including the following (Fig. 4):
  • Acute hepatitis, cirrhosis, fatty liver, drug-induced liver disease, obstruction of biliary flow by carcinoma at the head of the pancreas, bile duct stricture, primary biliary cirrhosis, and metastatic carcinoma of the liver.
 
Biliary Isoenzyme
Biliary isoenzyme or macrohepatic isoenzyme, is seen in the α1 position. It has been isolated in cases of metastatic carcinoma to the liver and has been suggested as a diagnostic tool in identifying such cases. It has also been isolated in patients with viral hepatitis, alcoholic cirrhosis and other liver diseases (Fig. 5).
Fig. 4: Liver cirrhosis alkaline phosphatase (ALP) isoenzyme pattern
235
Fig. 5: Biliary obstruction alkaline phosphatase (ALP) isoenzyme pattern
 
Bone Isoenzyme
The bone isoenzyme is seen in the β1 position. The β1 bone ALP elevated as a result of increased osteoblastic activity and it is normally elevated in growing children. The highest total ALP values have been attributed to an increased bone isoenzyme level due to Paget's disease or renal rickets. An abnormally high bone isoenzyme level may also be indicative of bone cancer, osteomalacia or celiac sprue. A decreased bone ALP in children may be attributed to cretinism or hypophosphatasia (Fig. 6).
 
Placental Isoenzyme
It appears in the serum of pregnant women late in the first trimester of pregnancy and may remain elevated for 1 month after termination of pregnancy. Infarction of the placenta in toxemia increases the serum placental isoenzyme.236
Fig. 6: Hyperosteoblastic activity alkaline phosphatase (ALP) isoenzyme pattern
 
Intestinal Isoenzyme
The intestinal isoenzyme is seen in the β2 position. It is normally seen in the serum of subjects who have B or O blood types, especially after a fatty meal. Pathologically, the band may be present in perforation of the bowel, ulcerative diseases of the intestine and faintly in liver cirrhosis, as well as in intestinal perforation.
 
Other Rare ALP Isoenzyme
 
Renal Isoenzyme
It is a rare isoenzyme reported by Nerenberg and Kranc which, like the Regan isoenzyme, migrates to the placental position. This isoenzyme represents a diseased state of the kidneys or rejection of kidney transplant.
 
Regan Isoenzyme
It is isolated from the sera of patients with neoplasms. Because of the similarities to placental isoenzyme, it has been referred to as carcinoplacental 237isoenzyme. Regan isoenzyme has been isolated from patients with lung cancer, breast cancer, ovarian cancer, and carcinoma of the colon.
 
Nagao Isoenzyme
It is a variant of Regan isoenzyme that migrates in the same position as Regan isoenzyme on cellulose acetate. It has been isolated in metastatic carcinoma to the pleural surfaces and in adenocarcinoma of the pancreas or bile duct.
 
Pa Isoenzyme
It is an unusual band observed in sera of patients with pancreatic cancer.
 
Artifact Band
It is a band migrating in the albumin position on cellulose acetate. Controversy exists as to the identification of this band. It may be an artifact caused by an albumin-bilirubin complex or by some other substance, or in some instances it may be a true isoenzyme. Further research must be conducted to determine its true origin and significance.

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266Index
Note: Page numbers followed by f refer to figure and t refer to table
A Abnormal serum protein electrophoresis, evaluation of Acanthocytosis in a patient with abetalipoproteinemia Acute and chronic inflammatory panel Acute attacks Acute disseminated encephalomyelitis Acute phase reactant response Agar gel electrophoresis Agarose gel , Albumin , , Alkaline phosphatase (ALP) isoenzyme electrophoresis separated with a special treatment electrophoresis isoenzyme Alpha () , Alpha (α) macroglobulin Alpha globulins Alpha zone , Amido black (naphthol blue black) stains Amido black (naphthol blue black) structure Amyloidosis Analbuminemic Analbuminemic condition Anemia Antibody molecule Antitrypsin , Artifact band Asymptomatic (Smoldering) myeloma Autoimmunology Autosomal recessive pattern of inheritance in abetalipoproteinemia B B cell lineage and associated diseases Behçet's disease , Bence Jones protein , , , Beta zone Biclonal gammopathy Biclonal profile Biliary isoenzyme Biliary obstruction alkaline phosphatase (ALP) Bisalbuminemia , Blood collection procedure Blood tests Blood-brain barrier breakdown Bone destruction in myeloma, causes of Bone isoenzyme Bone marrow microenvironment, role in bone resorption Bone marrow sample Bromophenol blue stains Bromophenol blue structure C Capillary electrophoresis gel electrophoresis isoelectric focusing isotachophoresis , zone electrophoresis , CD Cellulose acetate electrophoresis Cerebral vasculitis Cerebrospinal fluid electrophoresis, clinical significance of Cerebrospinal fluid oligoclonal Cerebrospinal fluid proteins Ceruloplasmin , Chromogen Chronic cerebrospinal venous insufficiency Colorimetric assay of CR Congenital analbuminemia Coomassie dye stains C-reactive protein Cryofibrinogen immunofixation Cryoglobulin , Cryoglobulinemia IInd type “a” Sec. brouet “b” Sec. brouet Cryoglobulinemic immunocomplexes Cryoprecipitate composition Cryoprecipitation mechanism D Densitometer scan of serum protein electrophoresis Destaining solution , Devic's disease Diffuse or disseminated syndromes Diseases biomarkers Double α globulin band E Electrophoresis methods for types of Electrophoretic phenotyping and quantification of CR Endogenous pathway Exogenous pathway Expanded disability status scale (EDSS) F Fibrinogen Fibrinogen band and absence of fibrinogen Fibrinolysis (simplified) Fluorescent dye stains Foix-Alajouanine syndrome Frictional resistance Functional group-specific Stains G Gammaglobulin zone, changes Gangrene and type I cryoglobulinemia Gel-to-gel reproducibility Globulin zone, changes Glomerular function Glomerulus and the tubule Gram stain H Haptoglobin Heterozygosis antitrypsin High density lipoprotein metabolism Hp phenotypes Human immunoglobulin G subclasses Human leukocyte antigen region of chromosome Hyperalbuminemia Hyperchylomicronemia , , Hyperlipoproteinemia primary , secondary Hyperosteoblastic activity alkaline phosphatase (ALP) isoenzyme pattern Hypertransferrinemia Hypoalbuminemia Hypogammaglobulin and normal gammaglobulin Hypogammaglobulinemia , , I IgA IgA κ monoclonal in α region IgG and IgM oligoclonal with Igs polyclonal type IIb CR IgG and IgM polyclonal type III CR IgG-κ monoclonal type I CR Immunglobulin A structure as a dimer Immunofixation Immunofixation electrophoresis , Immunoglobulin A , Immunoglobulin G , Immunoglobulin light chain Immunoglobulin M , Infectious diseases Inherited disorders Interferences in CR analysis Intermittent proteinuria Intestinal isoenzyme Intratest , Isoelectric focusing electrophoresis Isoelectric point L Lactate dehydrogenase Lectin Lipoprotein (a) or Lp (a) (Apo B) atherosclerosis electrophoresis in disease metabolism metabolism very low density patterns interpretation of measurement of Liver cirrhosis alkaline phosphatase (ALP), isoenzyme pattern Liver isoenzyme Low density lipoprotein metabolism Lumbar puncture M Macroglossia Malignant monoclonal gammopathies Mesangial cells , , , Microglobulin Microheterogene Migratio rate of Monoclonal band adjacent to C area central area polyclonal base medium conserved >γ area of which strongly retromigrated with depressed polyclonal base gammopathy , undetermined significance unknown significance immunoglobulin structures Multiple myeloma bone lesions Multiple sclerosis management of the effects N Nagao isoenzyme Native polyacrylamide gel electrophoresis Neisseria gonorrhoeae Neoplastic disorders Nephrotic syndrome , Nerve axon with myelin sheath Neurological symptoms Neuromyelitis optica Neurosarcoidosis Nigrosin stains Nonaqueous capillary electrophoresis O Oligoclonal Oligoclonal gammopathy Oligoclonal profile P Pa isoenzyme Paper electrophoresis apparatus Pentameric immunoglobulin M Plasmodium falciparum malaria Polyacrylamide gel electrophoresis structure Polyclonal gammopathy , hyperglobulinemia structure of the immunoglobulin , Ponceau S stains , Prealbumin , Prealbumin band Protein electrophoresis , Protein gel stains Protein stains for electrophoresis procedures Proteinuria Proteinurias of plasmatic derivation tubular derivation Q Quantitation of protein zones albumin area interalbumin a antitrypsin area , stains for visualization of serum R Reabsorption, mechanisms of Reagents and materials , Red blood cell count Regan isoenzyme Renal failure Renal isoenzyme Rheumatoid arthritis Rheumatoid factor S Sample collection and storage Semiquantitative detection of CR by cryocrit Serum protein electrophoresis clinical significance components of indications introduction typical normal pattern Serum sample analyzed on immunofixation electrophoresis agarose gel Silver stains Sjogren's syndrome , Sodium dodecyl sulfate polyacrylamide gel electrophoresis Starch gel electrophoresis Sudan black stain Syndromes autoimmune lymphoproliferative carpal tunnel chronic active EBV infection Crash CREST syndrome Fanconi Guillain-Barré , hyper IgE Lambert-Eaton myelodysplastic nephrotic , , , , , non-disseminated demyelination periodic fever Sjögren's , , tropical splenomegaly Wiskott Aldrich , Syphilis serology Systemic lupus erythematosus , T Transferrin , Triclonal gammopathy Tubular function Tubular proteinuria Two-dimensional electrophoresis U Ulcerative colitis Unrelated inflammatory disorders Urinary protein of a glomerular and tubular origin , Urinary proteins Urine tests V Values , Vascular disease Visual inspection Visual screening and qualitative detection of CR W Waldenström's macroglobulinemia Wash solutions Whipple's disease White blood cell count Z Zinc stains , Zone alpha alpha alpha beta electrophoresis gamma , principles of ,