Electrophoresis
• Principle:
– The movement of charged molecules in an electric
field
– The rate of migration is dependent on the net electrical
charge of the molecule, size, and shape of the
molecule, buffer pH, properties of the support medium,
time frame for the procedure and temperature of the
operating system
• Uses: Hgb electrophoresis, serum protein
electrophoresis
Cover
–
+
Power
supply
Anode
Insulating plate
Buffer
Cathode
Electrophoresis support medium
In an electrophoresis system, charged molecules move through a support medium
because of forces exerted by an electric field.
Albumin
a1
a2
b
g
Migration distance
This serum protein electrophoresis demonstrates a normal pattern, with the largest
peak for albumin.
Normal
Normal values may vary from lab to lab.
Serum protein electrophoresis
Total serum
protein
Amount in grams
per deciliter (g/dL)
Albumin:
58%–74%
3.5–5.5
Alpha-1
globulin:
2.0%–3.5%
0.2–0.4
Alpha-2
globulin:
5.4%–10.6%
0.5–0.9
Beta
globulin:
7%–14%
0.6–1.1
Gamma
globulin:
8%–18%
0.7–1.7
Principali componenti delle frazioni
elettroforetiche I
• Albumin. In addition to carrying substances through the
bloodstream, albumin proteins help keep fluid from
leaking out of tiny blood vessels (capillaries). Albumin
may also help with tissue growth and healing. More than
half of the protein in blood serum is albumin.
• Alpha-1 globulin. High-density lipoprotein (HDL), the
“good” type of cholesterol, is included in this fraction.
• Alpha-2 globulin. A protein called haptoglobin, that binds
with hemoglobin, is included in the alpha-2 globulin
fraction.
Principali componenti delle frazioni
elettroforetiche II
• Beta globulin. In addition to carrying substances through
the bloodstream, beta globulin proteins help fight
infection. One protein (transferrin) included in this
fraction helps carry iron in the blood. Another protein
(complement C3) is needed to destroy bacteria.
• Gamma globulin. These proteins are important in
preventing and fighting infection. Some gamma globulins
bind to foreign substances (such as bacteria or viruses),
causing them to be destroyed by the immune system.
(See an illustration of the immune system.) Gamma
globulins are also called antibodies.
High values
• An increase in the percentage of albumin can indicate a severe loss of
water from the bloodstream (dehydration).
• An increase in the percentage of alpha-1 globulin may be caused
bysystemic lupus erythematosus, rheumatoid arthritis, pregnancy,
infection, or cancer.
• An increase in the percentage of alpha-2 globulin may be caused by
systemic lupus erythematosus, rheumatoid arthritis, cancer, kidney
disease, heart attack, or a long-term (chronic) infection (such as
tuberculosis).
• An increase in the percentage of beta globulin may be caused by liver
disease (such as cirrhosis), anemia, or an increased lipid level.
• An increase in the percentage of gamma globulin may be caused by a
chronic infection, anautoimmune disease, some types of leukemia,
multiple myeloma, Waldenstrom's macroglobulinemia, liver disease,
or Hodgkin's disease. An increase can also occur normally with aging
(a condition called monoclonal gammopathy of unknown
significance).
Low values
• A decrease in the percentage of albumin may be caused by a poor diet
(malnutrition), severe burns, liver or kidney disease, systemic lupus
erythematosus, rheumatoid arthritis, Hodgkin's disease, cancer that
has spread in the body, or diseases (such assprue or Crohn's disease)
that prevent the intestines from absorbing the nutrients from food.
• A decrease in the percentage of alpha-1 globulin may be caused by
liver or kidney disease or by an inherited condition that can cause
emphysema.
• A decrease in the percentage of alpha-2 globulin may be caused by
liver damage, leukemia, hemolytic anemia, hypothyroidism, or
Wilson's disease.
• A decrease in the percentage of beta globulin may be caused by
kidney disease or problems with the blood-clotting process.
• A decrease in the percentage of gamma globulin may be caused by
kidney disease or problems of the immune system (low amount of
antibodies).
Laboratory investigations of liver disease
Serum protein electrophoresis
• Characteristic findings in liver
disease include reduction in
albumin and increase in
gamma globulin in chronic
liver disease (particularly
auto-immune chronic hepatitis
and alcoholic cirrhosis)
• Decrease in alpha-1 globulins
(alpha-1 antitrypsin
deficiency)
• Iincrease in alpha-globulins
(prolonged cholestasis)
•
SERUM PROTEIN ELECTROPHORESIS (SPE)
Albumin alpha1 alpha2 beta gamma
Pt’s SPE
NOTE: Instead of the normal polyclonal
distribution, Ig is distributed in a
monoclonal fashion. This is called a
“monoclonal gammopathy.” but first, we
must provide that this “band of restricted
mobility” is really a monoclonal antibody.
SPE tracing
TO PROVE THAT THE “BAND OF RESTRICTED
MOBILITY” IS TRULY A MONOCLONAL ANTIBODY, WE MUST PERFORM IMMUNOFIXATION
ELECTROPHORESIS (IFE).
IFE:
- run the pt’s serum in 6 lanes.
- In the first lane after electrophoresis, we precipitate all of the serum proteins with a polyclonal antiserum or chemical precipitation with
tricholoracetic acid (TCA). This pattern looks
like the normal SPE.
IMMUNOFIXATION ELECTROPHORESIS (IFE)
- In lanes 2 through 6, we overlay each lane with
specific antisera.
Lane
G
A
M
K
L
anti-serum
anti-gamma heavy chain
anti-alpha heavy chain
anti-mu heavy chain
anti-kappa light chain
anti-lambda light chain
Note: The antisera
precipitate the Ig’s
present in the lanes.
SP
G
A
M
K
L
-
+
anti-mu
anti-kappa
anti-lambda
SP
anti-alpha
poly-specific
anti-gamma
Normal Ig distribution: “polyclonal” pattern
G
A
M
K
L
-
Ag - Ab complexes “precipitate” in gel +
- other proteins in each lane are “washed” away
Polyclonal pattern
INTERPRETATION: The “M-spike” on the SPE by
IFE is composed of monoclonal IgG kappa