Serum Protein Electrophoresis PDF

Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 6...

Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 6 Dr. Hayder A. Serum protein electrophoresis Protein Structure The major structural and functional molecules produced by cells are proteins. They are important in host defense, cell structure, movement, and as regulatory molecules. Proteins are composed of individual units called amino acids. The term ‘electrophoresis’ refers to the migration of charged particles in an electrical field. The success of electrophoresis in separating serum, urine and cerebrospinal fluid proteins into clinically useful fractions results from the heterogeneity of the charges of these molecules. Cation : positively charged ion, it moves toward the cathode (-) Anion : negatively charged ion, it moves toward the anode (+) Amphoteric substance : can have a positive/negative/zero charge, it depends on conditions Principle: Some substances have different net charges and can be separated into several fractions in external electric field. But velocity of a particle also depends on the: size, shape of the particle and given applied voltage 1  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 6 Dr. Hayder A. 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 by amidoblack solution. Factors effecting migration of protein by electrophoresis 1-The charge of the particles 2-Applied of electric field 3-Temperature 4-Nature of suspended medium power supply (direct current) containers for staining and destaining gel electrophoresis chamber applicator 2  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 6 Dr. Hayder A. Serum protein electrophoresis Hydragel – agarose gel 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 Serum proteins are separated into 6 groups: Albumin α1 - globulins α2 - globulins β1 - globulins β2 - globulins γ - globulins 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. Electrophoretic pattern is constant under physiological conditions (intensity of bands). Spectrum of plasma proteins changes under various diseases (their ratio) evaluation of electrophoretic pattern (bands or peaks). Serum proteins electrophoresis in diagnostics of diseases 1- Normal pattern 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 3  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 6 Dr. Hayder A. 2- Acute inflammatory response α1 α2-globulins Immediate response occurs with stress or inflammation caused by infection, injury or surgical trauma normal or ↓ albumin ↑ α1 and α2 globulins 3- Chronic inflammatory response α1 α2 γ-globulins Late response is correlated with chronic infection (autoimmune diseases, chronic liver disease, chronic infection, cancer) normal or ↓ albumin ↑α1 or α2 globulins ↑↑ γ globulins 4- Liver damage – Cirrhosis γ-globulins Cirrhosis can be caused by chronic alcohol abuse or viral hepatitis ↓ albumin ↓ α1, α2 and β globulins ↑ Ig A in γ-fraction 4  Al-Bayan University 3ed stage College of Health and Medical Techniques adv. Lab. tech. Medical Laboratory Techniques Lec. 6 Dr. Hayder A. 5- Nephrotic syndrome α2-globulin β-globulin fractions 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) 6- Monoclonal gammopathy a sharp gamma globulin band 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. 5