2. Composition of Plasma
92% water
Proteins- for every 100ml for about 7.6 grams
Albumins, Globulins, Fibrinogen
http://www.joinbiomedics.com/bl
3. Plasma Protein Distribution
Other PlasmaProteins
(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, be ta and g am m a g lo bulin
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
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 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 low
Too much heating
Run at different pH
Use lower wattage or
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#
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
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
30.
31.
32.
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
34.
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
36.
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 α1 α2 β γ
“β-γ bridging”
38
39.
40.
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
43. Hypogammaglobulinemia
Albumin α1 α2 β γ
Decreased gamma globulins Condition Albumin
Globulins
α1 α 2 β γ
Hypogammaglo-
bulinemia
N N N N
M. Zaharna Clin. Chem. 2009
44.
45.
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
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
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