2. Determination of Hemoglobin in Whole Blood

3. 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.

4. 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.

5. Principle: In the presence of KCN and potassium ferrieyide [ Fe 4 (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.

6. 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.

7. 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

8. 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.

9. Clinical significance

10. Causes lead to increase in hemoglobin Primary this is may be caused by a gen aberration in hemocytoblastic cell in bone marrow that produce RBC S. or due to physiological polycythemia. Secondary due to hypoxia which cause the blood forming organ to produce large quantity of RBC S. 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 RBC S due to - Toxic factor. - Infection. - Radiation.