Serum protein electrophpresis

AGA KHAN UNIVERSITY HOSPITAL KARACHI

1. SERUM PROTEIN
ELECTROPHORESIS

2. Composition of Plasma
92% water
Proteins- for every 100ml for about 7.6 grams
Albumins, Globulins, Fibrinogen
http://www.joinbiomedics.com/bloodcells.gif

3. Plasma Protein Distribution
Other Plasma Proteins
(1%)
Fibrinogen
(4%)
Albumin
(60%)
Globulin
(35%)
Albumin (60%)
Globulin (35%)
Fibrinogen (4%)
Other Plasma
Proteins (1%)

4. 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

5. 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

6. Too Much Gamma Globulin
Protein?
You may have many diseases, including:
Chronic inflammatory disease
Hyperimmunization
Acute infection
Waldenstrom’s macroglobulinemia

7. 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

8. Other Plasma Proteins
remaining one 1% of plasma
Peptide hormones
Insulin
Prolactin
Glycoproteins
TSH (thyroid- simulating hormone)
FSH (follice stimulating hormone)
LH (luteinizing hormone)

9. Plasma Proteins Come From…
Liver
Synthesizes 90% of the proteins
Lymphocytes (lymphatic system)
Makes the plasma cells  antibodies
Endocrine organs
Peptide hormones

10. 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

11. 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

12. 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

13. 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)

14. Equipment used for the gel electrophoresis
in the practical training A1
power supply
(direct current)
electrophoresis
chamber
containers for staining
and destaining gel
applicator

15. COMMON PROBLEMS
Likely 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 sample
Precipitate in sample
Check sample for salt,
try different pH,
centrifuge of filter sample
first
Holes in staining patternAnalyte too high in
concentration
Problem
Very thin sharp bandsMW 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 low
Too much heating
Run at different pH
Use lower wattage or
external cooling

16. 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#

17. Globulin fractions
Alpha1 globulins:
alpha1 antitrypsin, alpha lipoproteins,
Alpha2 globulins:
caeruloplasmin,haptoglobins,alpha2 macroglobulin
Beta globulins:
- beta lipoprotein, transferrin, fibrinogen

Gamma globulins:
immunoglobulins, CRP

19. 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#

20. 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

21. 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

22. Serum proteins electrophoresis in diagnostics
of diseases
Normal pattern

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM
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

23. Hypoalbuminaemia
Haemodilution
Loss from the body
Acute phase
response
Decreased
synthesis
Pregnancy
Chronic illness

24. Haemodilution
Loss from the body
Acute phase response
Decreased synthesis
Pregnancy
Chronic illness

25. Alpha 1 antitrypsin
MW 50000
Protease inhibitor
Distributed in ECF
Increased in acute phase response
Decreased in inborn errors of metabolism or
nephrotic syndrome

26. Alpha2 macroglobulin
Large MW protein
MV 90000
Often increased in plasma in protein losing
states

27. Haptoglobins
Bind haemoglobin
Increased levels seen in acute phase
response
Decreased levels seen when there is
intravascular hemolysis or hemorrhage into
tissues

28. 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

29. Beta2 microglobulin
MW120000
Component of HLA complex found on
surfaces of all nucleated cells
Inceased levels in myeloma patients and
those with renal failure

33. 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

35. 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

37. 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

38. Hepatic cirrhosis
M. Zaharna Clin. Chem. 2009
Decreased albumin (synthesis)
Increased gamma globulins (polyclonal gammopathy)
Albumin a
1
a
2
b g
“b-g bridging”
38

41. 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

42. Nephrosis
Condition Albumin
Globulins
12βγ
Nephrosis N N
Albumin a
1
a
2
b g
Decreased albumin
Increased a
2
-macroglobulin
Decreased gamma globulins
42

43. Hypogammaglobulinemia
Albumin a
1
a
2
b g
Decreased gamma globulins
Condition Albumin
Globulins
α1α 2βγ
Hypogammaglo-
bulinemia
N N NN
M. Zaharna Clin. Chem. 2009

46. 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

47. Monoclonal gammopathy
M. Zaharna Clin. Chem. 2009
Albumin a
1
a
2
b g
Albumin decreased
Sharp peak in gamma region
47

51. 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

52. 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

53. 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

54. 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

55. 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

56. IgD
MW 190000
Less than 0.1 g/L are found in normal adults

57. 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

58. Causes of
hypergammaglobulinaemia
Polyclonal-diffuse increased intensity of
staining in the gamma globulin portion
Monoclonal-well demarcated band of protein
in the globulin area

59. Polyclonal
hypergammaglobulinaemia
Chronic liver disease
Chronic infections
Inflammatory disease of bowel
Autoimmune disorder
Granulomas

60. Monoclonal
hypergammaglobulinaemia
Benign
Idiopathic
Diabetes mellitus
Chronic infections
Cirrhosis
Connective tissue
disorders
Malignant
Multiple myeloma
Macroglobulinaemia

61. 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

62. 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

63. 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