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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 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
Dilute the serum and control with B-2 Barbital buffer (1 part sample:4 part buffer)
Dry
Dry
Dry
The pattern of the protein may then be visually interpreted or quantitated by densitometer
ALBUMIN
GLOBULIN
ALPHA ()
BETA ()
GAMMA()
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
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
Function:
Help prevent & fight infection
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
+
Albumin 1 2
Albumin 1
Monoclonal Gammopathy
Marked, single spike in gamma
+
Albumin 1 2
A potential to malignant
Polyclonal Gammopathy
- bridging
+
Albumin 1 2
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
Nephrotic Syndrome
+
Albumin 1 2
Nonspecific disorder Large amounts of protein leak out into the urine
Causes :
Edema
Analbuminemia
+
Albumin 1 2
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
monoclonal gammopathies and polyclonal gammopathies 2. Can be used to characterized diseases based on the patterns of proteins bands
If gel not stored in horizontal position, atypical electrophoretic patterns will be produced
Medicines such as corticosteroids, birth control pills& aspirin, will alter the results
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.