Clin. Chem 202
Electrophoresis and
its Application for
diagnosis
Ratchada Cressey, PhD
Principal of electrophoresis
• Electrophoresis is the process of moving
charged molecule in solution by applying an
electric field across the mixture
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Electrode
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Serum Protein Electrophoresis
(SPE)
• Electrophoresis is refer to
the migration of all
charged solutes or
particle in liquid medium
under influence of an
electric field
Serum Protein Electrophoresis
SPE procedure
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Sample preparation
Gel loading
Gel running
Protein staining
quantification
Procedure (i)
Membrane and Sample
preparation
Procedure (ii)
sample application and
electrophores
Separated protein bands can be
quantified using Densitometer
Densitometer is one of the
application of spectrophotometer
• Absorbance measurement is made through a solid
material,using support medium is the cuvet
• Accuracy and precision are limited by variations in
path length and scatter from the solid medium
Normal SPE
Albumin
Alpha-1 globulin
Alpha-2 globulin
Beta- globulin
Gamma globulin
47-71% (3.63-4.91 g/dL)
2.7-5.8% (0.11-0.35 g/dL)
5.1-12.0% (0.65-1.17 g/dL)
4.5-15.7% (0.74-1.26 g/dL)
11.3-24.0% (0.58-1.74 g/dL)
Clinical Significance of Albumin
• Reference range 3.5-5.5 g/dL
• Hyperalbuminemia
dehydration
• Hypoalbuminemia
• Low intake, synthesis
• Increase loss:
• kidney; nephrotic syndrome, wound, burn
• GI tract; protein-losing enteropathy
• Increase catabolism
Clinical Significance of a1-globulin
•Increased alpha 1 globulin due to increased
alpha 1 antitrypsin: inflammatory/acute phase
•Decreased/absent alpha1 globulin due to
alpha1antitrypsin deficiency: infant with
neonatal cirrhosis or child with emphysema
Clinical Significance of a2-globulin
•Increased alpha 2 globulin due to alpha 2
macroglobulin: nephrotic sx with peripheral
edema, membranous glomerulonephritis,
hyperlipidemia
•Decreased alpha 2 globulin due to
haptoglobin: hemolytic anemia with
spherocytes in PB
Clinical Significance of b-globulin
•Increased beta globulin due to increased
transferrin in iron deficiency anemia:
microcytic hypochromic RBCs; third trimester
of pregnancy
•Increased beta-gamma with bridging:
cirrhosis
Clinical Significance of g-globulin
MONOCLONAL GAMMOPATHY
• Monoclonal peak: narrow needle-shaped “spike”
• With plasma cell myeloma (IgG,IgA) or IgM type in
LPL lymphoplasmacytic lymphoma
• In gamma region usually IgG
• In gamma, beta or even alpha 2 globulin region,
usually IgA
• If at application point between beta & gamma
regions, IgM type
POLYCLONAL
HYPERGAMMAGLOBULINEMIA
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Caused by reactive processes
Broad asymmetric peak
Chronic infection including HIV+/AIDS
Autoimmune/collagen vascular disease:
systemic lupus erythematosus (SLE),
rheumatoid arthritis (RA)
• Liver disease
• The nephrotic pattern
Normal
• illustrates the long term loss of lower molecular weight proteins
(Examples: albumin, IgG) and retention of higher molecular
weight proteins (example alpha-2-macroglobulin)
• This patient was a 39 year old female with SLE. Her daily total
urinary protein loss exceeded 3700 mg. (Normal loss is less
that 150 mg/day.)
http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE
Normal
• Polyclonal gammopathy
• usually occurs secondary to many chronic diseases. This patient
was a 39 year old male with sarcordosis. The sequential increase of
the globulin fractions illustrated "sarcoid stepping." IFE excluded the
possibility of a monoclonal protein.
http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE
• Liver disease:
Normal
• Patient was a 46 year old male with end stage liver disease
secondary to chronic alcohol abuse. He died 5 months after
this sample was obtained.
• In the cirrhotic pattern, the distinction between beta and
gamma globulin is blurred and is sometimes referred to as the
"beta-gamma bridge" pattern.
http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE
Normal
• Acute inflammatory pattern
• This patient was a 42 year old female who presented with a
temperature of 40 degrees oC and was diagnosed with both
pneumonia and pyelonephritis.
• In the acute inflammatory pattern albumin and gamma globulin
are decreased and alpha-2-globulin becomes very prominent. In
both the strip and scan note that the intensity of the alpha-2
fraction is greater that the gamma globulin fraction.
http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE
Normal
• Alpha-1-anti-trypsin deficiency
• can be congenital or acquired, typically secondary to liver or
pulmonary diseases.
• Congenital alpha-1-anti-trypsin deficiency is most commonly
associated with early onset emphysema, pancreatic insufficiency,
or cirrhosis.
http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE
• Immunofixation (IFIX) is a powerful enhancement of
immunoelectrophoresis in which a series of postelectrophoretic gel slabs are layered with celluloseacetate gels saturated with specific antibodies.
• The resulting antigen-antibody complexes fixed on
the second gel may then be stained, allowing
sensitive and specific qualitative visual
identification of paraproteins by electrophoretic
position.
Immunofixation
Electrophoresis (IFE)
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Diluted patient serum is applied to each
track of the gel using an application mask.
The gel is electrophoresed, separating
proteins according to their charge.
Protein fixative and monospecific antisera
to IgG, IgA, IgM, Kappa and Lambda are
applied to the gel.
During incubation, insoluble immune
complexes are formed.
The gel is now washed, pressed, dried, and
stained.
Samples are examined for paraprotein
bands in the TSP and Ig tracks
IFE of serum
IFE of Urine
Samples required for IFE
• 100 mL aliquot of 24 hour urine
• No preservative required.
Random urine will be accepted, but not a
recommended specimen due to low protein content.
• 0.5 mL serum
• Monoclonalprotein present
• The patient was a 72 year old male
who presented with lower back
pain.
• Quantitative immunoglobulin
measurements showed a large
increase in serum IgG, but
decreased IgA and IgM. Bone
marrow exam revealed a large
increase in plasma cells that were
frequently aggregated.
• IFE on this patient's serum showed
the M protein was IgG kappa. A
diagnosis of multiple myeloma was
made.
http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE
• Biclonal gammopathy
• This sample is from a 62
year old male who
presented with weight loss
and fatigue. He was found to
have multiple myeloma.
• In this disease, biclonal
gammopathies are rare,
occurring in about 1.7 % of
patients. IFE showed the 2
M proteins to be IgG-k and
IgA-lambda
http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE
Electrophoresis of Urine
• a minimum of 10 ml of urine for
electrophoresis.
• Most urine samples require concentration to
obtain this minimum amount and for optimal
concentration,
• Electrophoresis on urine is the preferred
technique for identification of Bence Jones
proteins in patients with suspected multiple
myeloma.
• It should always be done in conjunction with
serum, so the results can be compared
directly.
Electrophoresis of Urine
Normal Range
Urine Total Protein
( < 140 mg/24hr )
Urine Albumin
( < 30 mg/24hr )