Estimation of haemoglobin

 

Estimation of haemoglobin by Sahli’s Method

 Aim:  determination of haemoglobin by using Sahli’s  method

  Principal:   when blood is added to 20.0 1 n HCL hemoglobin is converted into brown   coloured acid haematin forms.  the resulting colour after dilation should compared with standard brown a reference tube of Sahli’s haemoglobinometer

  Requirement

1. Sahli’s haemoglobinometer.

2. Two Pasteur pipettes (one for HCl and one for distilled water).

3. Glass rod to stir ( stirrer )

N - Hydrochloric acid

5. Distilled Water

6. Comparison tube.

7. Pipette ( Hemoglobin pipette with rubber tubing and mouthpiece )

 

Procedure.

1.     Add 0.1N.hydrochloric acid (1: 10 diluted) to the haemometer tube (comparison tube) up to lowest graduation ( 0.02 gramme ).

2.     Sterilize the fingertip with Isopropyl alcohol, surgical spirit and allow it to dry

3.     Using sterile lancet prick the finger tip4. Wipe away first few drops of blood5. Suck blood into the hemoglobin pipette ( capillary pipette ) up to 20 cu.mm( avoid air bubbles coming into a tube )

4.      Wipe the outside tip of pipette, clean with tissue

Cont. Procedure

1.     Immediately transfer the blood to the comparison
tube8. Suck blood back into the pipette several times and blow out again into the tube(to mix blood with HCl )

2.     Place the haemometer tube in the stand and allow for 5-10 minutes (during this period HCl lysis red cell, and released hemoglobin on reacting with HCl forms a dark brown colored Acid Hematin ).

3.     Now using a Pasteur pipette to add a few drops of distilled water (or 0.1N HCl) and stir the contents with a glass rod .

Cont. Procedure

1.     Continue to add water (or acid) drop by drop and stir the contents each time until the solution is just darker than the standard .

2.     Carefully add one or two drops of water till the color exactly matches with that of the Standard and note the reading

3.     while taking the reading hold the haemoglobinometer against good daylight at arms length

4.     The comparison tube represent 100 percent haemoglobin with reference to a standard which is g Hb / 100 ml of blood

Result:      In given blood sample  ---------- gm of Hb per 100 ml.