Estimation of Hemoglobin Concentration
Dr. R.K.Choudhary
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 betaglobulin 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.
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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
Sahli’s acid haematin and alkalinhaematin method.
• Haemoglobin concentration provides
information about the status of anaemia in
the population.
Normal adult Hb:
15 ± 2 g/dl
 Women 13.5 ± 1.5g/dl
 At birth 18 ± 4g/dl
 Men
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.
Principle
• 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.
• Hermann Sahli (May 23, 1856 – April 28, 1933) was
a Swiss internist.
Materials used
• 1. Sahli’s haemoglobinometer.
• 2. Two Pasteur pipettes (one for HCl and one for
distilled water).
• 3. Glass rod to stir ( stirrer )
• 4. 0.1 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 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
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 .
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 14. 8 g Hb / 100 ml of
blood
Cont. Procedure
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 .