1. Estimation of Hemoglobin Concentration
Dr. R.K.Choudhary

2. Description
Hemoglobin is the protein molecule in red blood cells that carries oxygen from the lungs to the body's tissues and returns carbon dioxide from the tissues back to the lungs.
Hemoglobin is made up of four protein molecules (globulin chains) that are connected together.
The normal adult hemoglobin (Hbg) molecule contains two alpha-globulin chains and two beta- globulin chains.
In fetuses and infants, beta chains are not common and the hemoglobin molecule is made up of two alpha chains and two gamma chains. As the infant grows, the gamma chains are gradually replaced by beta chains, forming the adult hemoglobin structure.

3. Methods available for Hb estimation
Sahli’s Method
Colorimetric Method.
Autoanalyzer.
Hemoglobin and Hematocrit (HCT) Test Meter Kit Sahli’s method were compared to Haemiglobincyanide (HiCN) method of same subjects.
Results: Sahli’s method is less accurate and has lower values than Haemiglobincyanide method

4. Sahli’s acid haematin and alkalin-haematin method.
Haemoglobin concentration provides information about the status of anaemia in the population.
Normal adult Hb:
Men 15 ± 2 g/dl
Women 13.5 ± 1.5g/dl
At birth 18 ± 4g/dl

5. Objective
• Hemoglobin is measured to detect anemia and its severity and to monitor anemic patient under the treatment.
• It is also used to check the Hb level of potential donor’s blood, prior of donation.

6. Hemoglobin is converted to acid hematin by the action of HCL
Hemoglobin is converted to acid hematin by the action of HCL. The acid hematin solution is further diluted until its colour matches exactly with that of the permanent standard of the comparator block. The Hb concentration read directly from the calibration tube.
Principle

7. Hermann Sahli (May 23, 1856 – April 28, 1933) was a Swiss internist.

8. Materials used 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 )

9. 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 tip
4. Wipe away first few drops of blood
5. Suck blood into the hemoglobin pipette ( capillary pipette ) up to 20 cu.mm( avoid air bubbles coming into a tube )
6. Wipe the outside tip of pipette, clean with tissue

10. Cont. Procedure 7. Immediately transfer the blood to the comparison
tube
8. Suck blood back into the pipette several times and blow out again into the tube(to mix blood with HCl )
9. 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 ).
10. 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 .

11. Cont. Procedure
11. Continue to add water (or acid) drop by drop and stir the contents each time until the solution is just darker than the standard .
12. Carefully add one or two drops of water till the color exactly matches with that of the Standard and note the reading
13.while taking the reading hold the haemoglobinometer against good daylight at arms length
14. The comparison tube represent 100 percent haemoglobin with reference to a standard which is g Hb / 100 ml of blood

12. Cont. Procedure

13. Cont. Procedure 7. Immediately transfer the blood to the comparison
tube
8. Suck blood back into the pipette several times and blow out again into the tube(to mix blood with HCl )
9. 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 ).
10. 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 .