Protein Electrophoresis PDF

PROTEIN Electrophoresis Dr. Fatima Chamseddine, MD Clinical Pathology Introduction Examination of the proteins in plasma can provide information reflecting disease states in many different organ systems. The most frequently performed measurement—that for total protein—is usuall...

PROTEIN Electrophoresis Dr. Fatima Chamseddine, MD Clinical Pathology Introduction Examination of the proteins in plasma can provide information reflecting disease states in many different organ systems. The most frequently performed measurement—that for total protein—is usually performed on serum. Although total protein determination gives the physician some information as to a patient’s general status regarding nutrition or severe organ disease (as in protein-losing states), further fractionations yield far more clinically useful information. Structure The primary structure of a protein is its linear sequence of amino acids with different side groups, which determine how the protein folds on itself (secondary and tertiary structures) and how it reacts with other molecules and cells (i.e., its molecular identity). Methods Methods to quantitate and fractionate proteins are based on turbidimetry, colorimetry, absorption spectrophotometry, dye binding, column chromatography, electrophoresis, and immunoassays  A colorimetric technique highly specific for proteins and peptides is the Biuret method, by which copper salts in alkaline solution form a purple complex with substances containing two or more peptide bonds. Interferences are minimal, although ammonium ion may acidify the reaction, while hemoglobin and bilirubin absorb in the same region as the Biuret complex (540 to 560 nm). The Biuret method is extensively used in clinical laboratories, particularly in automated analyzers in which protein concentration can be measured down to 10 or 15 mg/dL.  Nephelometry detects the turbidity produced usually within minutes or less by the precipitation of a reagent antibody with its target protein in a serum sample The major serum proteins are now widely measured by this method on automated immunochemistry analyzers Total Serum Protein (TSP): Albumin, Globulin & A/G ratio Albumin, low: Globulins, increased: Decreased synthesis: As part of acute or chronic Malnutrition reactions to disease Malabsorption Liver failure Lowering of albumin and Increased loss elevation of globulins tend to Proteinuria occur simultaneously in disease, Accumulation of ascites thus leading to exaggerated Enteropathy changes in the A/G ratio Protein electrophoresis Most proteins bear a net negative charge at pH 8.6, causing attraction to the anode (positive pole), but a counteractive force, endosmosis, may pull them towards the cathode. In the case of γ globulins, which have a weak net negative charge, the endosmotic force prevails and they are moved towards the cathode Capillary zone electrophoresis is similar in many respects to gel electrophoresis but is capable of greater resolution  Protein electrophoresis separates proteins according to their electrical charges (usually at pH 8.6) The major clinical use of serum and urine protein electrophoresis is to screen for monoclonal gammopathies  Patterns of protein electrophoresis in serum and urine are characteristic of specific diseases primarily involving changes in synthetic rates (liver), loss (renal), or inflammatory states. Immunofixation & immunotyping Immunofixation electrophoresis (IFE), immunotyping, and immunoelectrophoresis (IEP) are methods for characterizing a suspected monoclonal band IEP is no longer in routine clinical use IFE involves placing patient serum into each of 6 wells in a gel. After electrophoresis, 5 different antisera are applied to the gel: anti-IgG, IgA, IgM, k, and L Immunotyping (IT, immunosubtraction) is often used in conjunction with capillary electrophoresis  The major proteins in plasma that contribute to the electrophoretic pattern are albumin, α1-antitrypsin, α2- macroglobulin, haptoglobin, β- lipoprotein, transferrin, complement C3, fibrinogen, and immunoglobulins.  Several minor components of plasma proteins, such as ceruloplasmin, C-reactive protein, prealbumin, and protease inhibitors, have clinical utility in diagnosing and monitoring diseases and are quantitated by immunoassays. Hereditary deficiency of some plasma proteins leads to significant diseases (e.g., α1-antitrypsin). Proteins in plasma play several roles, including maintaining oncotic pressure, transporting small molecules, and promoting or inhibiting inflammatory reactions. Major serum proteins: 1- Albumin The most abundant serum protein. Synthesized in the liver It allows the clinician to interpret high or low Ca and Mg levels, because albumin binds about one-half of each of those ions Several alleles exist, the most common of which is albumin A Congenital absence (analbuminemia) characterized by mild edema and compensatory hyperlipidemia Clinical utility: assessment of hepatic synthetic function & nutritional status, Marked reductions in nephrotic syndrome, Negative acute phase reactant Half-life of albumin is 17 days 2- Prealbumin Not normally seen on SPEP but migrates faster than albumin Binds ~10% of circulating thyroxine (it is also called transthyretin [TTR] and thyroxine binding prealbumin) and binds retinol binding protein:vitamin A complex Clinical utility in the assessment of nutritional status Half-life: 48 hours 3- Alpha 1-antitrypsin (AAT) The major component of the α1- band Main function is protease inactivation AAT deficiency may be detectable on SPEP Rise in response to acute inflammation 4- Alpha 1-acid glycoprotein Positive acute phase reactant and a major component of the increased α1-band in acute inflammatory states 5- Alpha 2-macroglobulin Relative 10-fold increase in nephrotic syndrome (large size precludes filtration) 6- Ceruloplasmin Functions in copper transport In addition to Wilson disease, ceruloplasmin is decreased in hepatic failure, malnutrition… Falsely normal or elevated ceruloplasmin may be seen in inflammatory states (is an acute phase reactant) 7- Haptoglobin Third major component of the α2-band Binds free hemoglobin; rapidly depleted in hemolysis Does not bind myoglobin Haptoglobin can be quantitated in terms of its hemoglobin-binding capacity or by immunologic means, especially nephelometry. Acute phase reactant 8- Transferrin The major β-globulin, Transports ferric (Fe 3+) iron Markedly increased in iron deficiency, but also increased in pregnancy and estrogen therapy in cases of severe iron deficiency, an elevated level can have the appearance of a paraprotein (pseudoparaproteinemia) Elevated with chronic inflammation (decreases in the early stages of an acute phase response) Modified when crosses blood-brain barrier to asialated transferrin; thus, a hallmark of CSF protein electrophoresis is a double transferrin peak In normal serum, transferrin ranges from 200 to 400 mg/dL, which is conveniently measured as iron-binding capacity (IBC) 9- Fibrinogen A β-globulin that, normally consumed in the formation of the clot, is not present in serum protein electrophoresis It appears in plasma electrophoresis as a distinct band between the β- and γ-globulins Incomplete clotting may lead to pseudoparaprotein Fibrinogen levels become elevated with the other acute phase reactants 10- C-Reactive Protein CRP Produced in the liver and migrates in γ region, where it can produce a small pseudoparaprotein A marker of systemic inflammation High sensitivity CRP (hsCRP) assays may indicate cardiovascular risk ACUTE PHASE REACTANTS The acute phase reactant proteins share the property of showing elevations in concentrations in response to stressful or inflammatory states that occur with infection, injury, surgery, trauma, or other tissue necrosis. They include AAT, α1-acid glycoprotein, haptoglobin, ceruloplasmin, fibrinogen, serum amyloid A protein, and CRP. Others are factor VIII, ferritin, lipoproteins, complement proteins, and immunoglobulins. The elevation of acute phase reactants is likely a response to the cytokines These proteins provide another dimension of quantitation that can be useful for monitoring the course of a patient by serial determinations ACUTE PHASE REACTANTS-CRP A generally useful acute phase reactant for monitoring response is CRP, which is the fastest-rising acute phase reactant and one that returns to normal quickly following successful therapies. CRP is frequently applied to the detection and preliminary classification of occult infections, because bacterial infections can stimulate much higher levels of CRP than viral ones. Assessing disease activity in autoimmune disorders Patterns in serum protein electrophoresis SPEP Normal serum Nearly invisible prealbumin band large albumin band small peaked α1 band a somewhat broad α2 band bimodal β broad γ Bisalbuminemia Bisalbuminemia is seen in heterozygotes for albumin alleles. No clinical consequence A1-antitrypsin (AAT) deficiency AAT deficiency can be detected with SPEP (though it is not a sensitive or specific assay), since AAT is the major component of the α1 band Nephrotic syndrome Massive loss of small serum proteins, particularly albumin Decrease of all bands, especially albumin, with the exception of the α2 band that contains α2 macroglobulin Acute inflammation Increased acute phase reactants lead to increases in α1 and α2 bands. Albumin is slightly decreased With prolonged inflammation the gammaglobulins are polyclonally increased β - γ bridging cirrhosis This is the hallmark of cirrhosis, primarily due to increased serum IgA Additional features include hypoalbuminemia and blunted α1 and α2 bands Monoclonal gammopathy Monoclonal gammopathy (paraproteinemia or M protein) refers to a homogenous immunoglobulin in the serum Prominent, discrete, dark band (M spike) in the γ region, sometimes in β or α2 Most commonly the result of a plasma cell neoplasm, but may be present in lymphoma Monoclonal gammopathy Most commonly the result of a plasma cell neoplasm, but may be present in lymphoma, especially lymphoplasmacytic lymphoma or chronic lymphocytic leukemia CLL The M protein is usually composed of a heavy and a light chains, sometimes a light chain only and rarely a heavy chain only ~10% of patients with myeloma have an SPEP showing only hypogammaglobulinemia. This is typical of patients with light chain only myelomas Monoclonal gammopathy Immunofixation or immunosubstraction is indicated to characterize the M protein Pseudo M spikes: Fibrinogen (incompletely clotted sample), Hemoglobin (hemolyzed sample), Elevated CRP, Elevated transferrin, Certain antibiotics and some radiocontrast agents, Very high levels of serum tumor markers M protein quantitation carried out by nephelometry (or turbidimetry) Serum free light chains: important both diagnostically and in the monitoring of disease Serum viscosity may be increased in patients with IgM or IgA paraproteins CSF protein electrophoresis Electrophoresis of normal CSF Relatively prominent prealbumin band Double β (transferrin) band These findings may be used to diagnose CSF leak High resolution CSF electrophoresis may be used to support a diagnosis of multiple sclerosis (oligoclonal bands) Urine protein electrophoresis Glomerular proteinuria pattern Prominent albumin, (and β band) Reflects increased protein permeability in glomerulonephritis Urine protein electrophoresis Tubular proteinuria pattern Small albumin band, with prominent α1 and β-bands Reflects impaired tubular reabsorption in tubulointerstitial injury Urine protein electrophoresis Overflow proteinuria pattern Most commonly this is a monoclonal light chain (Bence Jones proteinuria) Other possibilities include myoglobin and hemoglobin Cryoglobulinemia Cryoglobulinemia is a systemic immune complex disease characterized by palpable purpura, arthralgias, hepatosplenomegaly, lymphadenopathy, anemia, sensorineural deficits, and glomerulonephritis. Most patients are hypocomplementemic Cryoglobulins Cryoglobulins are immunoglobulins that precipitate reversibly at low temperature Once detected, cryoglobulins may be characterized by electrophoresis and immunofixation 3 types of cryoglobulin 1. Type I cryoglobulins are monoclonal immunoglobulins found in association with plasma cell or lymphoid neoplasm Mixed 1. cryoglobulinemia (types II & III) 2. Type II cryoglobulins are a mixture of a monoclonal IgM and polyclonal IgG. The IgM has rheumatoid factor activity (anti-IgG). This is the most common type of cryoglobulin 3. Type III cryoglobulins are a mixture of 2 polyclonal immunoglobulins, typically IgG and IgM Found in a variety of clinical conditions, most commonly hepatitis C virus infection, in addition to lymphoproliferative disorders, chronic infections, chronic liver diseases, and autoimmune diseases (SLE)