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