Download - Serum protein electrophpresis
SERUM PROTEIN ELECTROPHORESIS
Composition of Plasma
92% water Proteins- for every 100ml for about 7.6
grams Albumins, Globulins, Fibrinogen
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Plasma Protein Distribution
Other Plasma Proteins (1%)
Fibrinogen
(4%)
Albumin
(60%)
Globulin
(35%)
Albumin (60%)
Globulin (35%)
Fibrinogen (4%)
Other PlasmaProteins (1%)
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
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
Too Much Gamma Globulin Protein? You may have many diseases, including:
Chronic inflammatory disease Hyperimmunization Acute infection Waldenstrom’s macroglobulinemia
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
Other Plasma Proteins
remaining one 1% of plasma Peptide hormones
Insulin Prolactin
Glycoproteins TSH (thyroid- simulating hormone) FSH (follice 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 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
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
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)
Equipment used for the gel electrophoresisin the practical training A1
power supply (direct current)
electrophoresis chamber
containers for staining and destaining gel
applicator
COMMON PROBLEMSLikely 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 samplePrecipitate 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 lowToo much heating
Run at different pHUse lower wattage or external cooling
Serum protein electrophoresisHydragel – agarose gel
Serum proteins are separated into 6 groups: Albuminα1 - globulinsα2 - globulinsβ1 - globulinsβ2 - globulinsγ - globulins
Figure is found at http://www.sebia-usa.com/products/proteinBeta.html#
Globulin fractions
Alpha1 globulins: alpha1 antitrypsin, alpha lipoproteins,
Alpha2 globulins: caeruloplasmin,haptoglobins,alpha2
macroglobulin
Beta globulins: - beta lipoprotein, transferrin, fibrinogen
Gamma globulins:
immunoglobulins, CRP
Hydragel 15/30
• Gels with 15 or 30 wells (serum samples) are used in laboratories of clinical biochemistry.
• Electrophoresis is also used for separation ofisoenzymes,nucleic acids and immunoglobulins
Figure is found at http://www.sebia-usa.com/products/proteinBeta.html#
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
Evaluation of separated protein fractionsDensitometry
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
Serum proteins electrophoresis in diagnostics of diseasesNormal pattern
Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM
Reference ranges:
Total protein 6.0 – 8.0 g/dLAlbumin 3.5 – 5.0 g/dLα1-globulins 0.1 – 0.4 g/dLα2-globulins 0.4 – 1.3 g/dLβ-globulins 0.6 – 1.3 g/dLγ-globulins 0.6 – 1.5 g/dL
Hypoalbuminaemia
Haemodilution Loss from the
body Acute phase
response
Decreased synthesis
Pregnancy Chronic illness
HaemodilutionLoss from the bodyAcute phase responseDecreased synthesisPregnancyChronic 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
Large MW protein MV 90000 Often increased in plasma in protein
losing states
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
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
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
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
Hepatic cirrhosis
M. Zaharna Clin. Chem. 2009
Decreased albumin (synthesis)Increased gamma globulins (polyclonal gammopathy)
Albumin 1 2
“- bridging”
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 (↑↑ α2-macroglobulin and ↑β-globulin)
Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM
α2-globulin β-globulin fractions
Nephrosis
Condition AlbuminGlobulins
1 2 β γ
Nephrosis N N
Albumin 1 2
Decreased albuminIncreased 2-macroglobulinDecreased gamma globulins
42
Hypogammaglobulinemia
Albumin 1 2
Decreased gamma globulinsCondition Albumin
Globulins
α1 α 2 β γ
Hypogammaglo-bulinemia
N N N N
M. Zaharna Clin. Chem. 2009
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
Monoclonal gammopathy
M. Zaharna Clin. Chem. 2009
Albumin 1 2
Albumin decreasedSharp peak in gamma region
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
MW 190000 Less than 0.1 g/L are found in normal
adults
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
Causes of hypergammaglobulinaemia Polyclonal-diffuse increased intensity of
staining in the gamma globulin portion
Monoclonal-well demarcated band of protein in the globulin area
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
Malignant Multiple myeloma Macroglobulinae
mia
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
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
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