Serum protein electrophpresis

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AGA KHAN UNIVERSITY HOSPITAL KARACHI

AGA KHAN UNIVERSITY HOSPITAL KARACHI

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  • 1. SERUM PROTEIN ELECTROPHORESIS
  • 2. Composition of Plasma  92% water  Proteins- for every 100ml for about 7.6 grams  Albumins, Globulins, Fibrinogen http://www.joinbiomedics.com/bl
  • 3. Plasma Protein Distribution Other PlasmaProteins (1%) Fibrinogen (4%) Albumin (60%) Globulin (35%) Albumin (60%) Globulin (35%) Fibrinogen (4%) Other Plasma Proteins (1%)
  • 4.  This water-soluble protein is the most abundant of all the plasma proteins.  Serum Albumin is the albumin present in blood  Is produced in the liver  Maintains osmotic pressure of plasma Albumins
  • 5. Globulins  4 different kinds of globulins present in blood: alpha 1 + alpha 2, be ta and g am m a g lo bulin  are transport proteins.  also serve as substrates for forming other substances  Gamma globulin makes up the largest portion of globulin
  • 6. Too Much Gamma Globulin Protein?  You may have many diseases, including:  Chronic inflammatory disease  Hyperimmunization  Acute infection  Waldenstrom’s macroglobulinemia
  • 7. Fibrinogen  Plasma protein that functions in blood clotting  Synthesized in the liver  Proactive protein and is converted to fibrin in certain conditions  Can cause heart attacks and strokes if there is too much in the blood stream
  • 8. Other Plasma Proteins  remaining one 1% of plasma  Peptide hormones  Insulin  Prolactin  Glycoproteins  TSH (thyroid- simulating hormone)  FSH (follice stimulating hormone)  LH (luteinizing hormone)
  • 9. Plasma Proteins Come From…  Liver  Synthesizes 90% of the proteins  Lymphocytes (lymphatic system)  Makes the plasma cells  antibodies  Endocrine organs  Peptide hormones
  • 10. 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 separated proteins are fixed and stained
  • 11. Serum protein electrophoresis on agarose gel is a type of horizontal gel electrophoresis The figure was found at http://www.mun.ca/biology/desmid/brian/BIOL2250/Week_Three/electro4.jpg
  • 12. CLINICAL APPLICATION  SPEP  Quantitative analysis of specific serum proteins  Identification and quantitation of Hb and its subclasses  Identification of monoclonal proteins in serum & urine  Seperation & quantitation of major lipoprotein  Isoenzyme analysis: LDH, CK,AP  Western Blot  Southern Blot
  • 13. Process of electrophoresis  1. sample application  2. adjustment of voltage or current - DIRECT CURRENT ! (gel-electrophoresis about 70 - 100 volts)  3. separation time: minutes (e.g. gel-electrophoresis of serum proteins 30 min.)  4. electrophoresis in supporting medium: fixation, staining and destaining  5. evaluation:  qualitative (standards)  quantitative (densitometry)
  • 14. Equipment used for the gel electrophoresis in the practical training A1 power supply (direct current) electrophoresis chamber containers for staining and destaining gel applicator
  • 15. COMMON PROBLEMS Likely cause Corrective Action No migration Instrument not connected Check electrical circuits Bowed electrophoretic pattern Overheating or drying out of support Check buffer ionic strength, reduce wattage Tailing of bands Salt in sample Precipitate in sample Check sample for salt, try different pH, centrifuge of filter sample first Holes in staining pattern Analyte too high in concentration Problem Very thin sharp bands MW of sample very high Use support larger pore size Very slow migration High MW, Low charge, Ionic strength too high, voltage too low Change pH, Check conductivity, dilute buffer, Increase voltage Sample precipitates in support pH too high or low Too much heating Run at different pH Use lower wattage or
  • 16. Serum protein electrophoresis Hydragel – agarose gel Serum proteins are separated into 6 groups: Albumin α1 - globulins α2 - globulins β1 - globulins β2 - globulins γ - globulins Figure is found at http://www.sebia-usa.com/products/proteinBeta.html#
  • 17. Globulin fractions Alpha1 globulins:  alpha1 antitrypsin, alpha lipoproteins, Alpha2 globulins:  caeruloplasmin,haptoglobins,alpha2 macroglobulin Beta globulins: - beta lipoprotein, transferrin, fibrinogen Gamma globulins:  immunoglobulins, CRP
  • 18. Hydragel 15/30 • Gels with 15 or 30 wells (serum samples) are used in laboratories of clinical biochemistry. • Electrophoresis is also used for separation of isoenzymes,nucleic acids and immunoglobulins Figure is found at http://www.sebia-usa.com/products/proteinBeta.html#
  • 19. Hydragel 15/30  Hypergamma Control Pictured  16-30 Figure is found at http://www.sebia-usa.com/products/proteinControl.html Normal Control Pictured 1-15
  • 20. Evaluation of separated protein fractions Densitometry  Densitometer is used for scanning of separated proteins in the gel. Scanning the pattern gives a quantitative information about protein fractions. Figure is found at http://www.aafg.org
  • 21. Serum proteins electrophoresis in diagnostics of diseases Normal pattern  Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM Reference ranges: Total protein 6.0 – 8.0 g/dL Albumin 3.5 – 5.0 g/dL α1-globulins 0.1 – 0.4 g/dL α2-globulins 0.4 – 1.3 g/dL β-globulins 0.6 – 1.3 g/dL γ-globulins 0.6 – 1.5 g/dL
  • 22. Hypoalbuminaemia  Haemodilution  Loss from the body  Acute phase response  Decreased synthesis  Pregnancy  Chronic illness
  • 23. Haemodilution Loss from the body Acute phase response Decreased synthesis Pregnancy Chronic illness
  • 24. Alpha 1 antitrypsin  MW 50000  Protease inhibitor  Distributed in ECF  Increased in acute phase response  Decreased in inborn errors of metabolism or nephrotic syndrome
  • 25. Alpha2 macroglobulin  Large MW protein  MV 90000  Often increased in plasma in protein losing states
  • 26. Haptoglobins  Bind haemoglobin  Increased levels seen in acute phase response  Decreased levels seen when there is intravascular hemolysis or hemorrhage into tissues
  • 27. Ceruloplasmin  Transfer protein for copper  Increased levels seen in acute phase response  Decreased levels seen in Wilsons Disease and malnutrition  Pregnant ladies and those on estrogen containing OCPs have increased levels
  • 28. Beta2 microglobulin  MW120000  Component of HLA complex found on surfaces of all nucleated cells  Inceased levels in myeloma patients and those with renal failure
  • 29. Acute inflammatory response • Immediate response occurs with stress or inflammation caused by infection, injury or surgical trauma • normal or albumin↓ • ↑ α1 and α2 globulins Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM α1 α2-globulins
  • 30. Chronic inflammatory response • Late response is correlated with chronic infection (autoimmune diseases, chronic liver disease, chronic infection, cancer) • normal or albumin↓ •↑α1 or α2 globulins •↑↑ γ globulins Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM α1 α2 γ-globulins
  • 31. Liver damage - Cirrhosis • Cirrhosis can be caused by chronic alcohol abuse or viral hepatitis • ↓ albumin • ↓ α1, α2 and β globulins • ↑ Ig A in γ-fraction Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM γ-globulins
  • 32. Hepatic cirrhosis M. Zaharna Clin. Chem. 2009 Decreased albumin (synthesis) Increased gamma globulins (polyclonal gammopathy) Albumin α1 α2 β γ “β-γ bridging” 38
  • 33. Nephrotic syndrome • the kidney damage illustrates the long term loss of lower molecular weight proteins (↓ albumin and IgG – they are filtered in kidney) • retention of higher molecular weight proteins (↑↑ α2-macroglobulin and ↑β-globulin) Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM α2-globulin β-globulin fractions
  • 34. Nephrosis Condition Albumin Globulins 1 2 β γ Nephrosis N N Albumin α1 α2 β γ Decreased albumin Increased α2-macroglobulin Decreased gamma globulins 42
  • 35. Hypogammaglobulinemia Albumin α1 α2 β γ Decreased gamma globulins Condition Albumin Globulins α1 α 2 β γ Hypogammaglo- bulinemia N N N N M. Zaharna Clin. Chem. 2009
  • 36. Monoclonal gammopathy  Monoclonal gammapathy is caused by  monoclonal proliferation of β-lymphocytal  clones. These „altered“ β-cells produce an  abnormal immunoglobulin paraprotein.  Production of paraprotein is associated  with benign monoclonal gammopathy  (leucemia) and multiple myeloma.  Paraproteins can be found in a  different position: between α-2 and  γ-fraction. Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM a sharp gamma globulin band
  • 37. Monoclonal gammopathy M. Zaharna Clin. Chem. 2009 Albumin α1 α2 β γ Albumin decreased Sharp peak in gamma region 47
  • 38. Immunoglobulins  Comprise the body's antibodies  Also involved in hypersensitivity reactions  Found in plasma gamma globulin fraction  Occasionally found in alpha2 and beta globulin fraction  Produced by B lymphocytes or mature plasma cells
  • 39. IgG  MW 160000  Protects extravascular tissue spaces  Made in response to soluble antigens  Transferred to baby from mothers blood across the placenta  Adult levels reached by 3-5 yrs of age
  • 40. IgA  Circulating IgA MW 160000  Secretory MW 400000  Protects body surfaces  Made in lamina propria of intestinal and laminal tracts  Levels low at birth  Reach adult levels by 15 yrs of age
  • 41. IgM  MW 900000  Protects the blood stream against foreign antigens  Foetus can synthesize IgM but levels are low at birth  High levels at birth indicate intrauterine infection  Adult levels are reached by nine months
  • 42. IgE  MW 200000  Involved in hypersensitivity reactions  Produced by plasma cells in respiratory tract, IT and nasopharynx  Bound to surface of mast cells and basophils  Adult levels are reached by 15 yrs of age
  • 43. IgD  MW 190000  Less than 0.1 g/L are found in normal adults
  • 44. Causes of hypogammaglobulinaemia  Decreased synthesis- transient (prematurity,3-6 mths olds) primary (IgA deficiency, genetic deficiency) Secondary (myeloma,CLL,DM,immunosuppressive drugs)  Protein loss- skin burns ,exudative lesions, protein losing enteropathy,nephrotic syndrome
  • 45. Causes of hypergammaglobulinaemia  Polyclonal-diffuse increased intensity of staining in the gamma globulin portion  Monoclonal-well demarcated band of protein in the globulin area
  • 46. Polyclonal hypergammaglobulinaemia  Chronic liver disease  Chronic infections  Inflammatory disease of bowel  Autoimmune disorder  Granulomas
  • 47. Monoclonal hypergammaglobulinaemia  Benign  Idiopathic  Diabetes mellitus  Chronic infections  Cirrhosis  Connective tissue disorders  Malignant  Multiple myeloma  Macroglobulinaemia
  • 48. Benign monoclonal hypergammaglobulinaemia  Serum paraprotein concentration of less than 20g/L (less than 10g/L if the paraprotein is an IgA)  Normal serum albumin  Present for five yrs or more without increase in paraprotein  Elderly
  • 49. Malignant monoclonal hypergammaglobulinaemia  Paraprotein concentration greater than 20g/L and increasing with time  Immune paresis (suppression of activity of other plasma cells)  Bence Jones proteins in urine  Characteristic bone marrow and X-ray findings
  • 50. SERUM IMMUNOFIXATION (IFE)  SPEP is a useful initial procedure to screen for an M- protein, but has two drawbacks  It is not as sensitive when M-proteins are small. An M- protein may be easily overlooked or an apparent M- protein may actually represent a polyclonal increase in immunoglobulins or another protein  If an M-protein is present, the immunoglobulin heavy and light chain class cannot be determined from the SPEP  Consequently, the lab must perform serum IFE in order to ascertain the presence of an M-protein and to determine its type