SERUM PROTEIN ELECTROPHORESIS

Composition of Plasma

92% water Proteins- for every 100ml for about 7.6 grams
 Albumins,

Globulins, Fibrinogen

http://www.joinbiomedics.co m/bloodcells.gif

Plasma Protein Distribution
Fibrinogen Other PlasmaProteins (4%) (1%)

Albumin (60%)

Globulin (35%)
Globulin (35%) Albumin (60%)

Fibrinogen (4%)

Other Plasma Proteins (1%)

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

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
 Insulin  Prolactin

hormones

 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 hormones

Endocrine organs
 Peptide

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 electrophoresis in the practical training A1
power supply (direct current)

containers for staining and destaining gel

electrophoresis chamber

applicator

COMMON PROBLEMS
Likely cause No migration Instrument not connected Corrective Action Check electrical circuits

Bowed electrophoretic pattern
Tailing of bands

Overheating or drying out of support
Salt in sample Precipitate in sample

Check buffer ionic strength, reduce wattage
Check sample for salt, try different pH, centrifuge of filter sample first Problem Use support larger pore size Change pH, Check conductivity, dilute buffer, Increase voltage Run at different pH Use lower wattage or external cooling

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

Sample precipitates in support

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#

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 of isoenzymes,nucleic acids and immunoglobulins

Figure is found at http://www.sebia-usa.com/products/proteinBeta.html#

Hydragel 15/30

 

Hypergamma Control Pictured 16-30

Normal Control Pictured 1-15

Figure is found at http://www.sebia-usa.com/products/proteinControl.html

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

Serum proteins electrophoresis in diagnostics of diseases Normal pattern

Reference ranges: Total protein Albumin α1-globulins α2-globulins β-globulins γ-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

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

Hypoalbuminaemia
  

Haemodilution Loss from the body Acute phase response

 

Decreased synthesis Pregnancy Chronic illness

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


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

α1 α2-globulins

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

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

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

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

γ-globulins

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

Hepatic cirrhosis
Decreased albumin (synthesis) Increased gamma globulins (polyclonal gammopathy)

“- bridging”

Albumin

1

2


M. Zaharna Clin. Chem. 2009


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)

α2-globulin β-globulin fractions

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

Nephrosis
Condition
Nephrosis

Albumin

Globulins 1 N 2 β N Decreased albumin Increased 2-macroglobulin Decreased gamma globulins γ

Albumin

1

2


42

Hypogammaglobulinemia
Decreased gamma globulins
Globulins Condition Hypogammaglobulinemia Albumin α1 N α2 N β N γ N

Albumin

1

2

M. Zaharna Clin. Chem. 2009

Monoclonal gammopathy
   

Monoclonal gammapathy is caused by a sharp gamma globulin band 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

Monoclonal gammopathy
Albumin decreased Sharp peak in gamma region

Albumin

1

2


47

M. Zaharna Clin. Chem. 2009

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 synthesistransient (prematurity,3-6 mths olds) primary (IgA deficiency, genetic deficiency) Secondary (myeloma,CLL,DM,immunosuppressive drugs)

Protein lossskin 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 Macroglobulinaemia

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 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

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