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A process of separating 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 but lacks fibrinogen and other substances that are used in coagulation process

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.

SPE is performed to: a) Screen for disease such as multiple myeloma, Waldenstroms macroglobulinemia and amyloidosis b) Find the causes of hypogammaglobulinemia (eg:Chronic Lymphocytic Leukaemia)

Multiple myeloma

Waldenstroms macroglobulinemia

Kidney amyloidosis

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 balance is known as isoelectric point (pI)

NEGATIVE

POSITIVE

The rate of migration of protein is dependent on:


a) b) c) d) e)

The net electrical charge of the molecule The size and shape of the molecule The electric field strength The nature of the supporting medium The temperature of operation

In brief, electrophoretic mobility is directly proportional to the net charge and inversely proportional to size of the molecule and the viscosity of the electrophoresis medium

- speed of separation

-has lower affinity for protein - native clarity after drying allow excellent densitometric examination

(20 minutes to 1 hour) - ability to store the transparent membranes for long periods

Used for electrophoretic separation of proteins in human serum, cerebrospinal fluid and urine It separates the human serum into five distinct, well-resolved zones

Blood is collected from patient

Dilute the serum and control with B-2 Barbital buffer (1 part sample:4 part buffer)

3-5l of diluted sample loaded into each template slot

Run electrophoresis for 25 minutes (votage:100V)

Acid solution I (3 minutes)

Dry

Paragon Blue Stain (3 minutes)


Dry

Dry

Acetic Acid 5% Solution I (2 minutes)

Acid Alcohol Solution II


(2 minutes)

Dry

Acetic Acid 5% Solution II


(2 minutes)

The pattern of the protein may then be visually interpreted or quantitated by densitometer

ALBUMIN

GLOBULIN

ALPHA ()

BETA ()

GAMMA()

The largest protein component of human serum It functions:

To keep the blood from leaking out of blood vessel Carry some medicine & other substances

Moving toward the negative portion of the gel Involve of 1 & 2 components

1 is comprised of : 1-antitrypsin
Thyroid binding globulin Trancortin - HDL also include in this fraction

2 consist of: Ceruloplasmin 2-macroglobulin Haptoglobin- binds to hemoglobin

Has 2 peaks:
1 2

Function:
Help carry substances, e.g: iron

Fight infection

Beta1
- Composed mostly of transferrin

Beta2
- Contains -lipoprotein - IgA, IgM & sometimes IgG along with complement also can be identified in this fraction

Most of clinical interest focused in this fraction Contains immunoglobulin (antibodies)

Function:
Help prevent & fight infection

Total serum protein


Albumin

Amount in grams per deciliter (g/dL)

Amount in SI units grams per liter (g/L)

58%74%

3.55.5

3555

Alpha-1 globulin
Alpha-2 globulin Beta globulin Gamma globulin

2.0%3.5%
5.4%10.6%

0.20.4
0.50.9

24
59

7%14%
8%18%

0.61.1
0.71.7

611
717

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

CLINICAL INTERPRETATIONS OF

SERUM PROTEIN ELECTROPHORESIS

Normal SPE pattern


Direction of migration

+
Albumin 1 2

Albumin 1

Monoclonal Gammopathy
Marked, single spike in gamma

+
Albumin 1 2

Diseases : Multiple myeloma Waldenstroms macroglobulinemia Amyloidosis Plasma cell leukemia

Characterized by proliferation of a single clone of plasma cells

A potential to malignant

Polyclonal Gammopathy
- bridging

A broad diffuse band

+
Albumin 1 2

Diseases : Viral infection (Hepatitis,HIV) SLE Hepatic cirrhosis Rheumatoid arthritis

Characterized by proliferation of more than one clone of plasma cells


Caused by any reactive or inflammatory process Associated with non-malignant condition

Hypogammaglobulinemia
Decreased Gamma region

+
Albumin 1 2

Primary : Bruton disease Specific antibody deficiency (SAD) Secondary Protein-losing enteropathy Immunosuppressive therapy

Condition which the level of immunoglobulins in blood is low Increased risk of infection

Gamma region almost absent

Nephrotic Syndrome

+
Albumin 1 2

Nonspecific disorder Large amounts of protein leak out into the urine

Decrease in most serum proteins Marked increases of alpha 2 fraction

Causes :
Edema

Analbuminemia

+
Albumin 1 2

Absence of albumin from the blood (Hypoalbuminemia)

Marked decreased in albumin region Increased alpha2, beta and gamma region.

However, in a particular disorder, the serum electrophoretic will show normal pattern. Therefore, in these cases, we need to use the CSF or urine instead of serum.
Detection of Bence Jones Protein in Urine Detection of oligoclonal band in CSF

Indication : Multiple myeloma Amyloidosis B-cell lymphoproliferative

Indication : Multiple sclerosis

1. Can be used to differentiate

monoclonal gammopathies and polyclonal gammopathies 2. Can be used to characterized diseases based on the patterns of proteins bands

Haemolysed blood will increased the globin value

If gel not stored in horizontal position, atypical electrophoretic patterns will be produced

Patient undergoing chemotherapy is not suitable to take this test

Lipaemic samples are not recommended for analysis

Cannot be used for patient that pregnant

Medicines such as corticosteroids, birth control pills& aspirin, will alter the results

1. Serum Protein Electrophoresis is

applicable for screening of particular disease based on the patterns of protein bands. 2. As for monoclonal gammopathies patients, further evaluation such as immunofixation is required to determine the specific causes of the abnormality.

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