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Composition of Plasma
92% water Proteins- for every 100ml for about 7.6 grams
Albumins,
Globulins, Fibrinogen
http://www.joinbiomedics.co m/bloodcells.gif
Albumin (60%)
Globulin (35%)
Globulin (35%) Albumin (60%)
Fibrinogen (4%)
Albumins
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
Globulins
4 different kinds of globulins present in blood: alpha 1 + alpha 2, beta and gamma globulin are transport proteins. also serve as substrates for forming other substances Gamma globulin makes up the largest portion of globulin
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
hormones
Glycoproteins
TSH
Liver
Synthesizes
Endocrine organs
Peptide
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
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)
electrophoresis chamber
applicator
COMMON PROBLEMS
Likely cause No migration Instrument not connected Corrective Action Check electrical circuits
Holes in staining pattern Very thin sharp bands Very slow migration
Analyte too high in concentration MW of sample very high High MW, Low charge, Ionic strength too high, voltage too low pH too high or low Too much heating
Globulin fractions
Alpha1 globulins: alpha1 antitrypsin, alpha lipoproteins, Alpha2 globulins: caeruloplasmin,haptoglobins,alpha2 macroglobulin Beta globulins: - beta lipoprotein, transferrin, fibrinogen
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
Hydragel 15/30
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
Reference ranges: Total protein Albumin 1-globulins 2-globulins -globulins -globulins 6.0 8.0 g/dL 3.5 5.0 g/dL 0.1 0.4 g/dL 0.4 1.3 g/dL 0.6 1.3 g/dL 0.6 1.5 g/dL
Hypoalbuminaemia
Haemodilution Loss from the body Acute phase response Decreased synthesis Pregnancy Chronic illness
Alpha 1 antitrypsin
MW 50000 Protease inhibitor Distributed in ECF Increased in acute phase response Decreased in inborn errors of metabolism or nephrotic syndrome
Alpha2 macroglobulin
Haptoglobins
Bind haemoglobin Increased levels seen in acute phase response Decreased levels seen when there is intravascular hemolysis or hemorrhage into tissues
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
Beta2 microglobulin
MW120000 Component of HLA complex found on surfaces of all nucleated cells Inceased levels in myeloma patients and those with renal failure
Immediate response occurs with stress or inflammation caused by infection, injury or surgical trauma normal or albumin 1 and 2 globulins
1 2-globulins
Late response is correlated with chronic infection (autoimmune diseases, chronic liver disease, chronic infection, cancer) normal or albumin 1 or 2 globulins globulins
Cirrhosis can be caused by chronic alcohol abuse or viral hepatitis albumin 1, 2 and globulins Ig A in -fraction
-globulins
Hepatic cirrhosis
Decreased albumin (synthesis) Increased gamma globulins (polyclonal gammopathy)
- bridging
Albumin
38
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 ( 2-macroglobulin and -globulin)
Nephrosis
Condition
Nephrosis
Albumin
Albumin
42
Hypogammaglobulinemia
Decreased gamma globulins
Globulins Condition Hypogammaglobulinemia Albumin 1 N 2 N N N
Albumin
Monoclonal gammopathy
Monoclonal gammapathy is caused by a sharp gamma globulin band monoclonal proliferation of -lymphocytal clones. These altered -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 -2 and -fraction.
Monoclonal gammopathy
Albumin decreased Sharp peak in gamma region
Albumin
47
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
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
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
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
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
IgD
Causes of hypogammaglobulinaemia
Decreased synthesistransient (prematurity,3-6 mths olds) primary (IgA deficiency, genetic deficiency) Secondary (myeloma,CLL,DM,immunosuppressive drugs)
Causes of hypergammaglobulinaemia
Polyclonal hypergammaglobulinaemia
Chronic liver disease Chronic infections Inflammatory disease of bowel Autoimmune disorder Granulomas
Monoclonal hypergammaglobulinaemia
Benign Idiopathic Diabetes mellitus Chronic infections Cirrhosis Connective tissue disorders
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
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
SPEP is a useful initial procedure to screen for an Mprotein, but has two drawbacks It is not as sensitive when M-proteins are small. An Mprotein may be easily overlooked or an apparent Mprotein 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