2021923 serum protein electrophoresis
TRANSCRIPT
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A process ofseparating electrically
charged particles in solution by passing
an electric current through the solution
Fluid that is separated from clotted blood
Similar in composition to plasma butlacks fibrinogen and othersubstances that are used incoagulation process
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Used an electric field to separate the
proteins in the blood serum into groups of
similar size, shape and charge.
Particularly used to determine
whether the humoral immunity
function normally or not.
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SPE is performed to:
a) Screen for disease such as multiple myeloma,Waldenstroms macroglobulinemia andamyloidosis
b) Find the causes ofhypogammaglobulinemia(eg:Chronic Lymphocytic Leukaemia)
Multiple myeloma Waldenstromsmacroglobulinemia
Kidney amyloidosis
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Based on the heterogeneity of charges of the protein molecules
Overall charge of protein molecule is determined by pH of its
solvent
pH at which the positive
and negative charges of
a given protein balanceis known as isoelectric
point (pI)
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POSITIVE
NEG
ATIVE
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The rate ofmigration of protein is dependent on:
a) The net electrical charge of the molecule
b) The size and shape of the moleculec) The electric field strength
d) The nature of the supporting medium
e) The temperature of operation
In brief, electrophoretic mobility is directly proportional to
the net chargeand inversely proportional to size of the
molecule and theviscosityof the electrophoresis medium
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- speed of separation(20 minutes to 1 hour)
- ability to store thetransparent membranesfor long periods
-has lower affinityforprotein
- native clarity afterdryingallow excellent
densitometricexamination
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Used for electrophoretic separation of proteins in
human serum, cerebrospinal fluid and urine
It separates the human serum into five distinct,
well-resolved zones
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Blood is collectedfrom patient
Dilute the serumand control with
B-2 Barbital buffer
(1 part sample:4part buffer)
3-5l of dilutedsample loaded
into each
template slot
Runelectrophoresisfor 25 minutes(votage:100V)
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Acid solution I
(3 minutes)
Paragon BlueStain
(3 minutes)
Acetic Acid 5%Solution I
(2 minutes)
Acid AlcoholSolution II
(2 minutes)
Acetic Acid 5%Solution II
(2 minutes)
Dry Dry
Dry
Dry
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The pattern of the
protein may then bevisually interpreted or
quantitated by
densitometer
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ALBUMIN GLOBULIN
ALPHA () BETA () GAMMA()
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The largest protein component
of human serum
It functions:To keep the blood from leaking out of blood
vessel Carry some medicine & other substances
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1 is comprised of : 1-antitrypsin
Thyroid binding globulin
Trancortin
- HDL also include in this fraction
2consist of:Ceruloplasmin
2-macroglobulin
Haptoglobin- binds to hemoglobin
Moving toward the negative
portion of the gel
Involve of1 & 2 components
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Most of clinical
interest focused in
this fraction
Contains immunoglobulin
(antibodies)
Function:
Help prevent & fight infection
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Total serum
protein
Amount in grams per
deciliter (g/dL)
Amount in SI units grams
per liter (g/L)
Albumin 58%74% 3.55.5 3555
Alpha-1globulin 2.0%3.5% 0.20.4 24
Alpha-2
globulin5.4%10.6% 0.50.9 59
Beta
globulin7%14% 0.61.1 611
Gamma
globulin8%18% 0.71.7 717
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HRE was introduced to replaced the traditional 5-band
electrophoretic methods (SPE)
The reason for this replacement is because SPE is less
sensitive since it can only produces 5 bands
HRE can produce
12 bands
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CLINICAL INTERPRETATIONS OF
SERUM PROTEINELECTROPHORESIS
N l SPE
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Normal SPE pattern
Albumin 1
2
+ -
Direction of migration
Albumin 1 2
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Monoclonal Gammopathy
Albumin 1
2
+ -
Characterized by
proliferation of a single cloneof plasma cells
Marked, single spike in
gamma
A potential to malignant
Diseases :Multiple myeloma
Waldenstroms
macroglobulinemia
Amyloidosis
Plasma cell leukemia
P l l l G h
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Polyclonal Gammopathy
Albumin 1
2
+ -
-bridging
Characterized by
proliferation of more thanone clone of plasma cells
Caused by any reactive or
inflammatory process
Associated with non-malignant
condition
A broad diffuse band
Diseases :
Viral infection(Hepatitis,HIV)
SLE
Hepatic cirrhosis
Rheumatoid arthritis
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Hypogammaglobulinemia
Albumin 1
2
+ -
Decreased Gamma
region
Condition which the level of
immunoglobulins in blood islow
Increased risk of infection
Gamma region almost absent
Primary :
Bruton diseaseSpecific antibody deficiency
(SAD)
Secondary
Protein-losing enteropathy
Immunosuppressive therapy
N h ti S d
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Nephrotic Syndrome
Albumin 1
2
+ -
Nonspecific disorder
Large amounts of protein leak
out into the urine
Causes :
Edema
Decrease in most serum proteinsMarked increases of alpha 2
fraction
A lb i i
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Analbuminemia
Albumin 1
2
+ -
Marked decreased in albuminregion
Increased alpha2, beta and gamma
region.
Absence of albumin from theblood (Hypoalbuminemia)
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However, in a particular disorder, the serum
electrophoretic will show normal pattern.
Therefore, in these cases, we need to use
the CSF orurine instead of serum.
Detection ofBence
Jones Protein in UrineDetection ofoligoclonal
band in CSF
Indication :Multiple myeloma
Amyloidosis
B-cell
lymphoproliferative
Indication :
Multiple sclerosis
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1. Can be used to differentiatemonoclonal gammopathies and
polyclonal gammopathies2. Can be used to characterized
diseases based on the patterns of
proteins bands
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Haemolysed blood willincreased
the globin value
Lipaemic samples arenot recommended for
analysis
Cannot be used forpatient that pregnant
If gel not stored inhorizontal position,
atypical electrophoreticpatterns willbe produced
Patient undergoingchemotherapy is not
suitable to takethis test
Medicines such ascorticosteroids,
birth control pills&aspirin,will alter the
results
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1. Serum Protein Electrophoresis isapplicable for screening of particulardisease based on the patterns of
protein bands.2. As for monoclonal gammopathies
patients, further evaluation such asimmunofixation is required to
determine the specific causes of theabnormality.