CCCS4 SPEP Respiratory HE A1ATD-1-41 PDF

Clinical chemistry case study Serum protein electrophoresis Respiratory system History and Examination A case study on “a1-antitrypsin deficiency” Introduction Plasma makes up 46-63% of blood Straw colored or clear Similar to interstitial fluid Differences: level...

Clinical chemistry case study Serum protein electrophoresis Respiratory system History and Examination A case study on “a1-antitrypsin deficiency” Introduction Plasma makes up 46-63% of blood Straw colored or clear Similar to interstitial fluid Differences: levels of respiratory gases Concentration and types of dissolved proteins Composition of Plasma: 92% water Proteins: for every 100ml for about 7.6 grams Albumins (60%), Globulins (35%), Fibrinogen (4%), and other plasma proteins (1%) Albumin This water-soluble protein is the most abundant of all the plasma proteins. Produced by the liver Maintains osmotic pressure of plasma Globulins 4 different kinds of globulins present in blood: alpha 1, alpha 2, beta and gamma globulin. Serve different functions including: transport substrates for forming other substances Gamma globulin: the largest portion of globulin, majority of gamma are immunoglobulins Fibrinogen Plasma protein that functions in blood clotting Synthesized in the liver Proactive protein and is converted to fibrin in certain conditions should be absent from serum protein electrophoresis Other Plasma Proteins Remaining one 1% of plasma Peptide hormone: examples: Insulin and Prolactin Glycoproteins: examples: TSH (thyroid- simulating hormone) FSH (follicle stimulating hormone) LH (luteinizing hormone) Plasma Proteins Come From… Liver Synthesizes 90% of the proteins Lymphocytes (lymphatic system) Makes the plasma cells  antibodies Endocrine organs Peptide hormones Serum Protein ElectroPhoresis (SPEP) Serum Protein Electrophoresis (SPEP) is lab technique performed to identify types of proteins present in the blood. Venous blood sample processed into serum proteins separated by size and charge (separated into 4 types seen from last slide) This technique is useful way to diagnose some diseases. Serum protein electrophoresis on agarose gel Principle: Serum proteins are negative charged at pH 8.6 (a buffer helps to maintain a constant pH) and they move toward the anode at the rate dependent on their net charge. The speed of running depends on the amount of net charge and molecular weight of the protein molecule The separated proteins are fixed and stained by an amino acid staining dye SPEP is a type of horizontal gel electrophoresis Serum protein electrophoresis Serum proteins electrophoresis in diagnostics of diseases Densitometry and Normal pattern A Densitometer is used for scanning of separated proteins in the gel. Scanning the pattern gives a quantitative information about protein fractions. (Scans are quantified using image processing software which can count the number of colored pixels in a region of the image) Major contents of each band Albumin Zone Albumin is the major fraction in a normal SPEP. A fall of 30% is necessary before the decrease shows on electrophoresis. The fastest migration towards the anode Usually a single band is seen. A decreased level of albumin, is common in many diseases, including liver disease, malnutrition, malabsorption, protein-losing nephropathy and enteropathy. Albumin - alpha-1 interzone High levels of Alpha fetoprotein (AFP) that may occur in hepatocellular carcinoma may result in a sharp band between the albumin and the alpha-1 zone. Alpha-1 zone Alpha 1 antitrypsin (A1AT) constitutes most of the alpha-1 band. Isolated band decrease is seen in the Alpha 1 antitrypsin deficiency state. The alpha-1 fraction does not disappear in alpha 1- antitrypsin deficiency because other proteins, including alpha-lipoprotein (HDL) and orosomucoid (alpha-1-acid glycoprotein, an acute phase reactant), also migrate there. Decrease combined with albumin decrease indicates protein loss (e.g. nephrotic syndrome), liver disease, malnutrition As a positive acute phase reactant, A1AT is increased in acute inflammation. Decreased Alpha 1 antitrypsin Decreased Alpha 1 antitrypsin Increased Alpha 1 antitrypsin Alpha-2 zone Consists principally of a-2 macroglobulin and haptoglobin. Haptoglobin: low levels in hemolytic anemia (haptoglobin is a suicide molecule which binds with free hemoglobin released from red blood cells and these complexes are rapidly removed by phagocytes). Haptoglobin is raised as part of the acute phase response, resulting in a typical elevation in the alpha-2 zone during inflammation. Increased Haptoglobin in acute inflammation Alpha-2 zone (contd.) a-2 macroglobulin (AMG) (720 Kda) elevations are seen as a sharp front to the alpha-2 band. AMG is markedly raised (10-fold increase or greater) in association with glomerular protein loss, as in nephrotic syndrome. Due to its large size, AMG cannot pass through glomeruli, while other lower-molecular weight proteins are lost. Enhanced synthesis of AMG accounts for its absolute increase in nephrotic syndrome. (hypothesized that this is a response to low albumin) Increased a-2 macroglobulin in nephrotic syndrome Nephrotic syndrome protein loss, several bands are decreased but: a2 band is increased is due to a-2 macroglobulin Beta zone Divided into Beta 1 and Beta 2 (normally Beta 1 > Beta 2) Beta 1 subzone Transferrin and beta-lipoprotein (LDL) Increased beta-1 protein due to the increased level of free transferrin is typical of iron deficiency anemia, pregnancy, and estrogen therapy. Increased beta-1 protein due to LDL elevation occurs in hypercholesterolemia. Decreased beta-1 protein occurs in acute or chronic inflammation. Increased b1 >> b2 in Iron deficiency anemia Beta zone Beta-2 subzone: comprised of Complement protein 3 and fibrinogen Complement protein 3 (C3). Increased in the acute phase response. Decreased in autoimmune disorders as the complement system is activated and the C3 becomes bound to immune complexes and removed from serum. Fibrinogen, found in normal plasma but absent in normal serum Occasionally, blood drawn from heparinized patients does not fully clot, resulting in a visible fibrinogen band between the beta and gamma globulins (SPEP should be done on a serum sample to stop fibrinogen interference) Gamma zone The immunoglobulins (Igs) are generally the only proteins present in the normal gamma region. Immunoglobulins consist of heavy chains, (A , M, G, E, and D and light chains (kappa and lambda). A normal gamma zone should appear as a smooth 'blush,' or smear, with no asymmetry or sharp peaks. The gamma globulins may be elevated, decreased, or have an abnormal peak or peaks. Note that Igs may be also be found in other zones; IgA typically migrates in the beta-gamma zone, and in particular, pathogenenic Igs may migrate anywhere, including the alpha regions. g Immunoglobulins structure 1. Antigen binding (Fab) region 2. Fragment crystallizable region (Fc) region 3. Heavy chain (blue) with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3) domains 4. Light chain (green) with one variable (VL) and one constant (CL) domain 5. Antigen binding site (paratope) 6. Hinge regions Summery of SPEP in different disease states Acute inflammatory response Immediate response occurs with stress or inflammation caused by infection, injury or surgical trauma normal or ↓ albumin ↑ α1 and α2 globulins Some proteins of the acute-phase response. A, albumin; CRP, C- reactive protein; HG, haptoglobin; T, transthyretin (previously called pre-albumin). The latter pair can be termed negative acute-phase proteins. From Candlish JK and Crook M. Notes on Clinical Biochemistry. Singapore: World Scientific Publishing, 1993. Chronic inflammatory response Late response is correlated with chronic infection, chronic inflammation, autoimmune diseases, and cancer normal or ↓ albumin ↑α1 or α2 globulins ↑↑ g globulins Liver damage - Cirrhosis Cirrhosis can be caused by e.g. chronic alcoholism or viral hepatitis ↓ albumin ↓ α1, α2 and β globulins Increased gamma globulins (polyclonal gammopathy) b-g bridging due to polyclonal increase in Ig A extending into b region Nephrotic syndrome The kidney damage illustrates the long term loss of lower molecular weight proteins (↓albumin and IgG –filtered in kidney glumerulus) and retention of higher molecular weight proteins (↑↑ α2- macroglobulin) β-globulin may be increased due to increased levels of LDL (not shown) Hypogammaglobulinemia Polyclonal Gammopathy Monoclonal gammopathy Monoclonal gammopathy (also called paraproteinemia) is the presence of excessive amounts of paraprotein or single monoclonal gamma globulin in the blood. It is usually due to an underlying immunoproliferative disorder or hematologic neoplasms, especially multiple myeloma. Multiple myeloma is caused by monoclonal proliferation of β-lymphocytal clones. These “altered” β-cells produce an abnormal immunoglobulin paraprotein. The increased protein has several deleterious effects on the body, including abnormally high blood viscosity and kidney damage. Monoclonal gammopathy Diagnosis These are characterized by the presence of any abnormal protein that is involved in the immune system, When a paraproteinemia is present in the blood, there will be a narrow band, or spike, in the serum protein electrophoresis because there will be an excess of production of one protein On SPEF, paraproteins can be found in different position: between α-2 and g-fraction. (keep this in mind as a differential for isolated increased protein peaks in other zones of SPEP) Monoclonal gammopathy Types Paraproteinemias may be categorized according to the type of monoclonal protein found in blood: Light chains only (or Bence Jones protein). This may be associated with multiple myeloma or Amyloid light-chain amyloidosis Heavy chains only (also known as "heavy chain disease"); Whole immunoglobulins. In this case, the paraprotein goes under the name of "M-protein" ("M" for monoclonal). Monoclonal Gammopathies Monoclonal gammopathy on SPEP Immunofixation electrophoresis Immunofixation electrophoresis (IFE) can be used to further identify abnormal bands, in order to determine which type of immunoglobulin is present. Principle When a soluble antigen (in this case heavy or light immunoglobulin chains) is brought in contact with the corresponding antibody, precipitation occurs. Washing the excess antibodies and other proteins that are not precipitated guarantees that only the targets are fixed to the electrophoresis gel which may be visualized by staining. Monoclonal protein detection by IFE A 72 year old male who presented with lower back pain. Quantitative immunoglobulin measurements showed a large increase in serum IgG, but decreased IgA and IgM. Bone marrow exam revealed a large increase in plasma cells that were frequently aggregated. IFE on this patient's serum showed the M protein was IgG kappa. A diagnosis of multiple myeloma was made. Immunofixation electrophoresis Results interpretation Where positive precipitation reaction occurred indicates the type of the immunoglobulin. (See previous slide) Polyclonal immunoglobulins appear as diffuse bands while a monoclonal immunoglobulin appears as a narrow band IFE technique can be also applied to detect proteins other than immunoglobulins or immunoglobulin derivatives. In this case specific antibodies against those proteins are used. (See the example of fibrinogen band identification in the next slide)