Determining Haemoglobin In Blood | Pysiology Practical

Haemoglobinometry: Determination of Haemoglobin in the Blood.

Theory:

Hemoglobin (Hb) is the iron containing coloring matter of red blood cell (RBC). It is a chromoprotein forming 95% of dry weight of RBC and 30% to 34% of wet weight. Function of hemoglobin is to carry the respiratory gases, oxygen and carbon dioxide. It also acts as a buffer. Molecular weight of hemoglobin is 68,000.

STRUCTURE OF HEMOGLOBIN

Hemoglobin is a conjugated protein. It consists of a protein combined with an iron containing pigment.
The protein part is globin and the iron containing pigment is heme. Heme also forms a part of the structure of myoglobin (oxygen binding pigment in muscles) and neuroglobin(oxygen binding pigment in brain).

IRON

Normally, it is present in ferrous (Fe2+) form. It is in unstable or loose form. In some abnormal conditions, the iron is converted into ferric (Fe3+) state, which is a stable form.

PORPHYRIN

The pigment part of heme is called porphyrin. It is formed by four pyrrole rings (tetrapyrrole) called, I, II, III and IV. The pyrrole rings are attached to one another by methane (CH4) bridges. The iron is attached to ‘N’ of each pyrrole ring and ‘N’ of globin molecule.

GLOBIN

Globin contains four polypeptide chains. Among the four polypeptide chains, two are alpha chains and two are beta chains
Hemoglobin is of two types:
1. Adult hemoglobin – HbA
2. Fetal hemoglobin – HbF


Haemoglobin Determination By Sahlis Method.

The underlying principal of this method is simple. Red cell membrane is destroyed by the addition of N/10 Hcl. Haemoglobin releases into plasma, reacts with the acid and forms acid hematin, which is brownish color compound. The solution is diluted by adding distilled water drop wise till the color matches with the standard color provided in the sahlis apparatus.

Requirements:

Sahlishaemoglobinometer, sterilized lancet, Micropipette, N/10 Hcl, distilled water and cotton soaked in alcohol.

Procedure:

  1. Place 5 drops of N/10 Hcl in the bottom of graduated sahlis tube.
  2. With the help of lancet prick the tip of your finger.
  3. Collect big drop of blood and suck blood upto mark 20 (0.02 ml) with out any air bubble.
  4. Insert the tip of Pipette in sahlis tube beneath surface of HCl, gently blow out the blood.
  5. Mix the blood and HCl with glass rod and wait for 10 min. the color of solution turns brownish which means acid hematin formation.
  6. Place the tube in between reference tubes in front of light.
  7. Now add distilled water drop by drop to acid hematin solution until its color matches with reference tubes.
  8. Read the reading on a tube to obtain gram of Hb per dl of blood.

Precautions:

      1. Carefully prick your finger.
      2. Always use a sterilized lancet
      3. Tip of micropipette must be disposable.
      4. Clean your laboratory after doing this lab work.
Results: The blood is taken in a test tube.To this blood 5 drops of HCl are added and acid hematin is produced.Then the tube is kept in sahlishemaglobinometer,water is added drop vise to the tube and compared with the refrencetubes.The color of subject blood sample matches with reference color at scale reading of 16 Hb/dl.