2021923 serum protein electrophoresis

7/28/2019 2021923 Serum Protein Electrophoresis

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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.

2021923 serum protein electrophoresis

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