Determination of
Hemoglobin in Whole
Blood
The iron atoms are linked to the four N atoms of
protoporphyrin by bonds which are all identical.
In hemoglobin, four hem units are linked to four
polypeptide chains, the product having a molecular
weight of approximately 68,000 Da. Combination of
one hem with a single polypeptide give the pigment,
myoglobin, present in muscle tissue. Hemoglobin and
myoglobin have the property of combining with oxygen
and carbon dioxide in a reversible fashion giving rise to
their important role as respiratory pigments .
There are a large number of genetic variants of
the polypeptide chains which combine with hemi
to produce abnormal hemoglobin some of which
show disordered function.
Many method used for determination of
blood hemoglobin,only the cyanmethoglobin
method has gained popular acceptance.
According to this technique, total hemoglobin at
alkaline pH is rapidly converted to the cyano –
derivative which is determined by its
absorbance at 540 nm.
Principle:
In the presence of KCN and potassium
ferrieyide [ Fe4 (CN)6 ] at alkaline media (pH) ,
hemoglobin and oxidized to methomoglobin,
which has maximum absorption at 540 nm . The
color in testy of this wavelength is proportional
to total heamoglobin concentration.
Procedure:
1 – Add 20 µl ( 0.02 ) of blood to 4 or
5.0 of reagent
2 – Stand at least 4 min and read
against a reagent blank at 540 nm .
3 – Read the standard at the same
way .
Calculation:
Blood hemoglobin ( g/100ml )
=Reading Au of unknown X D.F X conc. of standard
Reading As of standard
The D.F is 201 or 251according to whether 4.0 or
5.0 ml of reagent is used
1 – The hemoglobin value is less than the normal
value , the patient is said to be anemic
2 – The hemoglobin value is higher than the
normal value , the patient may have
polycythemia or erythrocytosis ( a higher than
normal concentration , hematocrit or
erythrocyte count ) usually provide an important
value to the likely causes of the anemia .
Clinical significance
Causes lead to increase in hemoglobin
•Primary this is may be caused by a gen aberration in hemocytoblastic cell in
bone marrow that produce RBCS . or due to physiological polycythemia .
•Secondary due to hypoxia which cause the blood forming organ to produce
large quantity of RBCS .
Causes that lead to decrease hemoglobin
•Decrease rate of production may be due to
•Destruction or loss the function of the blood forming tissue as in leukemia and
multiple myeloma.
•Luck of iron and protein in the food .
•Increase loss or destruction of RBCS due to
- Toxic factor.
- Infection .
- Radiation.