Transcript Hemoglobin (Hb

Hemoglobin Concentration
Determination
Hemopoiesis
Is the process of blood cell formation which takes
place during the embryonic life in the yolk sac;
mesenchyme and blood vessels; liver; spleen,
thymus and lymph nodes; bone marrow, while in
late fetus & adult takes place in bone marrow and
lymphtic tissues in normal situation (medullary
hemopoiesis).
 In pathological conditions hemopoiesis is
(extramedullary) in the liver, spleen and lymph
nodes.

Hemoglobin (Hb)
Hemoglobin (Hb) is the standard abbreviation for
hemoglobin, the oxygen-carrying pigment and predominant
protein in the red blood cells.
 Hemoglobin is the protein that carries oxygen from the
lungs to the tissues and carries carbon dioxide from the
tissues back to the lungs.
 In order to function most efficiently, hemoglobin needs to
bind to oxygen tightly in the oxygen-rich atmosphere of the
lungs and be able to release oxygen rapidly in the relatively
oxygen-poor environment of the tissues. It does this in a
most elegant and intricately coordinated way.
 Hemoglobin forms an unstable, reversible bond with
oxygen. In its oxygenated state it is called oxyhemoglobin
and is bright red. In the reduced state it is called
deoxyhemoglobin and is purple-blue.
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Structure of hemoglobin
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
A hemoglobin molecule consists
of four polypeptide chains: two
alpha chains, each with 141
amino acids and two beta chains,
each with 146 amino acids. The
protein portion of each of these
chains is called "globin".
The α and β globin chains are
very similar in structure and each
one of them is liked with a heme
molecule.
Structure of hemoglobin
Heme molecules
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A heme group is a flat ring
molecule containing carbon,
nitrogen and hydrogen atoms, with
a single Fe2+ ion at the center.
Without the iron, the ring is called a
porphyrin.
Changes in the amino acid
sequence of these chains results in
abnormal hemoglobin's. For
example, hemoglobin S is found in
sickle-cell disease, a severe type of
anemia in which the red cells
become sickle-shaped when oxygen
is in short supply.
Hemoglobin facts and stats
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Each RBC contains 280 million Hb molecules.
The average adult contains about 5 million
RBCs /mm3.
The average adult has about 5 litters of
blood.
Thus the average adult has about 790 gm of
Hb.
Normal Human Hemoglobin's
Embryonic
hemoglobin's
Fetal
hemoglobin
Adult hemoglobin's
gower
gower
Portland-
hemoglobin F
Hemoglobin A
hemoglobin A2
hemoglobin F
{2, 2}
{2, 2}
{2, 2}
>95%
<3.5%
1-2 %
Normal Ranges
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In the very common laboratory test for hemoglobin (Hb),
it is measured as total hemoglobin and the result is
expressed as the amount of hemoglobin in grams (gm)
per deciliter (dl) of whole blood, a deciliter being 100
milliliters.
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The normal ranges for hemoglobin depend on:
 The age.
 Altitude
 The sex of the person.
The normal ranges
Newborns
One (1) week of age
One (1) week of age
One (1) month of age
17-22gmdl
17-22gm\dl
15-20gmdl
11-15gm\dl
Children
Adult men
Adult women
11-13gm\dl
14-18gm\dl
12-16gm\dl
Men after middle age
Women after middle age
12.4-14.9gm\dl
11.7-13.8gm\dl
• Normal values in an adult are 12 to 18 grams per deciliter
(100 milliliters) of blood.

Above-normal hemoglobin levels is called
polycythemia which is may be:
 Secondary polycythemia which is may be due to:
 Dehydration (sever burns, diarrhea, vomitting,
…etc.).
 Severe lung or heart disease.
 Living at high altitudes.
 Heavy smoking.
 Primary polycythemia which is due malignant
variation in blood cells production in bone marrow

Below-normal hemoglobin levels may lead to anemia that
can be the result of:
 Iron deficiency or deficiencies in essential vitamins of
other elements, such as B12, folate, B6.
 Inherited hemoglobin defects, such as sickle cell anemia
or Thalassaemia.
 Other inherited defects affecting the red blood cells.
 Excessive bleeding.
 Excessive destruction of red blood cells.
 Kidney disease.
 Bone marrow failure or aplastic anemia.
 Cancers that affect the bone marrow.
Measurement of hemoglobin
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The Cyanmethemoglobin Method for Hb determination is
the reference method.
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Principle:
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Whole blood is diluted in a solution of potassium
Ferricyanide and potassium cyanide.
The Hb is oxidize to methemoglobin by the potassium
Ferricyanide.
The potassium cyanide then converts the methemoglobin
to cyanmethemoglobin.
The absorbance of the cyanmethemoglobin at 540 nm is
directly proportional to the Hb concentration.
Sulfhemoglobin is not converted to cyanmethemoglobin;
therefore, it can not be measured by this method.
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Hb (Fe++) K3Fe (CN)6 methemoglobin (Fe+++ )
KCN
Cyanmethemoglobin
Procedure of standard curve
1.
Create a standard curve, using a commercially
available cyanmethemoglobin standard which,
has constant concentration 25g/dl, the
following dilutions should be made to get the
line between the concentration & the
absorbance of the standard using also drabkin
reagent as shown:
Procedure
Hb
concentration
g/dl
Absorbance
reading
Volume of St /
ml
V of
Drabkin
reagent /
ml
0
0
0
5
5
0.125
1
4
10
0.250
2
3
15
0.375
3
2
7.5
0.188
2.5
2.5
Procedure
2.
3.
Transfer the dilutions to cuvettes. Starting with
the blank, measure the absorbance on a
spectrophotometer at 540 nm.
Plot absorbance on the y-axis and the Hb
concentration on the x-axis. The Hb
concentrations of the patients’ samples and
controls can be read from this standard curve.
Standard Curve
Absorbance•
0.500
0.375
0.250
0.125
0
5
10
15
Concentration
20
Calculation
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Slope = abs (st)
Con (st)
1
Slope
C (x)
C (x)
= Con (st) = Factor
abs (st)
= Con (st) X abs (x)
abs (st)
= Factor
X abs (x)