Transcript Hemoglobin Electrophoresis

o
Hemoglobin is a protein in red
blood cells that carries oxygen.
o
Each Hb molecule has a
complex quaternary shape.
o
It has two alpha chains and
two beta chains of polypeptides.
o
Each individual chain is a
globular protein subunit.
o
Each subunit contains a single
molecule of heme a ring
surrounding a single ion of iron.
Type of
Hemoglobin
Hemoglobin A
(HbA)
Polypeptide Chains
Notes
2 alpha, 2 beta
97% of normal
adult haemoglobin
Hemoglobin A2
(HbA2)
2 alpha , 2 delta
2.5% of normal
adult haemoglobin
Hemoglobin F
(HbF)
2 alpha , 2 gamma
Normal fetal
haemoglobin
This type of hemoglobin is present in sickle
cell disease.
This type of hemoglobin does not carry
oxygen well.
This type reduced synthesis of β chain may
cause β thalassemia.
This type of hemoglobin is present in a sickle
cell disorder.
This type of hemoglobin
may be present in certain types of thalassemia.


Hemoglobin S is responsible for most
types of sickle cell disease, a condition
in which the red blood cells have a
crescent or sickle shape .
It is caused by a point mutation in the
β-globin chain of haemoglobin, causing
the hydrophilic amino acid glutamic
acid to be replaced with the
hydrophobic amino acid valine at the
sixth position.

When sickle haemoglobin (HbS) gives up its oxygen to the
tissues, HbS sticks together .
◦ Forms long rods form inside RBC
◦ RBC become rigid, inflexible, and sickle-shaped
◦ Unable to squeeze through small blood vessels, instead
blocks small blood vessels
◦ Less oxygen to tissues of body
 2 copies of the gene
for Hb (each parent)
 HbS –Recessive
• S=Sickle
• A=Normal
- Sickle haemoglobin (HbS) + Sickle haemoglobin (HbS)
- Sickle haemoglobin (HbS) + (HbC)
- Sickle haemoglobin (HbS) + reduced HbA

1.
2.
3.
4.
5.
6.
Most people with sickle cell disease have at least
mild symptoms of chronic anemia, including:
Weakness and fatigue.
A pale appearance.
Yellowing of the skin and the whites of the eyes
(jaundice).
Shortness of breath.
Enlargement of spleen
Liver cirrhosis
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Hemoglobin S solubility test and Sodium Metabisulfite Test:
This test detects the presence of hemoglobin S but does not distinguish
between sickle cell disease and trait.
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Hemoglobinopathy (Hb) evaluation. :
- Hemoglobin electrophoresis the presence of various hemoglobins.
- Hemoglobin fractionation by HPLC
- Isoelectric focusing.
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o
o
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Complete blood count (CBC)
Blood smear
Reticulocytes
Iron test

In thalassemia the genetic defect, which could be either mutation ,
results in reduced rate of synthesis or no synthesis of one of the
globin chains that make up hemoglobin.

Severe forms of thalassemia are usually diagnosed in early
childhood and are lifelong conditions.
1.
2.
Alpha Thalassemia.
Beta Thalassemia

Four genes are involved in making the alpha globin part of
hemoglobin two from each parent. Alpha thalassemia occurs
when one or more of these genes is variant or missing.
1.
One gene affected
Two genes affected
Three genes affected
2.
3.
Silent Alpha Thalassemia
Alpha Thalassemia Trait (minor)
Hemoglobin H Disease

Two genes are involved in making the beta globin part of
hemoglobin one from each parent. Beta thalassemia occurs
when one or both of the two genes are variant.
1.
One gene is affected
Beta Thalassemia Trait (minor)
2.
Both genes are variant
Beta Thalassemia Intermedia
Beta Thalassemia Major
(Not Common)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Fatigue
Weakness
Shortness of breath
Pale appearance
Yellow discoloration of skin (jaundice)
Irritability
Facial bone deformities
Slow growth
Abdominal swelling
Dark urine
The signs and symptoms you experience depend on the type
and severity of thalassemia you have.
- WBC
- Nucleated RBC
- Microcytic Hypochromic
- Anisocytosis ( Schistocyte / Target cells )
- Reticulocytes
- Iron test
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It is a test that measures the different types of the
oxygen carrying protein (hemoglobin) in the blood.

Electrophoresis is a technique used to
separate and sometimes purify
macromolecules especially proteins
and nucleic acids that differ in size,
charge or conformation

When charged molecules are placed in
an electric field, they migrate toward
either the positive or negative pole
according to their charge. In contrast
to proteins, which can have either a net
positive ( NH3+) or net negative
charge ( COO- )
1.
2.
3.
4.
5.
6.
Strength of electric field
Size of molecules
Viscosity
Temperature
Ionic strength
Net charge

The blood sample must be drawn on the tube containing
EDTA anti clotting.

Wash is part of the sample (approximately from 500 to 700
microns) several times by saline solution (Normal Saline) and
in order to eliminate impurities and proteins found in the
sample.

Placed 200-400 microns of the sample in a special tube then
add the solution after crushing (haemoliyzed), which works to
break down red blood cells where hemoglobin is liberated
from.
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Processing the paper cellulose acetate and soaked it in the
buffer solution gradually from the top paper to bottom and
vice.

Making sure the completely immersing of the paper cellulose
acetate in a solution of buffer.

Then, 10 microns of sample processed are added to the pleats
in the paper of cellulose acetate by pipette.

Finally, the apparatus is hooked up to a power source under
appropriate running conditions to separate the protein bands.

The gel be stained by ponceaus to allowing visualization
of the separated proteins.
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After staining, different proteins will appear as distinct
bands within the gel.

It is common to run molecular weight size markers of
known molecular weight in a separate lane in the gel, in
order to calibrate the gel and determine the weight of
unknown proteins by comparing the distance traveled
relative to the marker.
A2/C
Normal
S
F
A+
A2/C
Normal
Hb SS
S
F
A+
A2/C
Normal
Hb SS
Hb AS
S
F
A+
A2/C
Normal
Hb SS
Hb AS
Hb SC
S
F
A+
A2/C
Normal
Hb SS
Hb AS
Hb SC
Hb CC
S
F
A+
A2/C
Normal
Hb SS
Hb AS
Hb SC
Hb CC
HB AD
S
F
A+
Normal
Sickle cell
Thalasemia
Hgb C
disease
HPFH
Hgb
A1
Hgb
A2
95-98%
2-3%
4-5.8%
Hgb
F
0.8-2.0%
( minor )
2-5%
5-35%
( minor )
Heterozygous
or
or
10-90%
100%
(Homozygous)
15%
under
2%
( major )
Hgb
S
Hgb
C
Hgb
H
0%
0%
0%
Hgb H
disease
70-98%
90-98%
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http://labtestsonline.org/understanding/analyte
s/hemoglobin-var/tab/glance
http://www.nlm.nih.gov/medlineplus/ency/articl
e/003639.htm
http://sickle.bwh.harvard.edu/thalover.html
http://www.as.miami.edu/chemistry/2086/chap
19/newchapter%2019-part1.htm
http://www.sicklecelldisease.org/index.phtml
http://en.wikipedia.org/wiki/Sickle-cell_disease
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