Haemoglobin Estimation
• Haemoglobin (Hb) is the main component of red blood cells and is a conjugated protein.
• A molecule of Hb contains two pairs of polypeptide chains α2β2 and four haem groups
each having an atom of ferrous ion.
• The oxygen carrying capacity of Hb when fully saturated is 1.34 ml/g.
• Approximately 34% of the RBCs by weight is Hb.
• Iron content of Hb is 0.347gm/100 g.
• The main function of Hb is to transport oxygen from lungs to the tissues.
• There are various forms of Hb as under:
i. Oxyhaemoglobin (HbO2)
ii. Carboxy haemoglobin (COHb)
iii. Sulfhaemoglobin (SulfHb)
iv. Methaemoglobin (MetHb)
• The measurement of concentration of Hb in the blood is known as haemoglobinometry.
Types of blood sample used for estimation of Hb are as follows:
a. Capillary blood from finger prick
b. Intravenous sample: It should be well anticoagulated, preferably in EDTA. Liquid
anticoagulants should not be used at all because they dilute and decrease Hb
concentration.
Methods for estimation of haemoglobin
Various methods used for the estimation of Hb are divided into four groups as:
I. Colorimetric method:
Colorimetric method is based on colorimetric measurement of the intensity of colour
developed on addition of some substances to the blood. Colorimetric methods include
the following;
1. Cyanmethaemoglobin method
2. Oxyhaemoglobin method
3. Electronic counter method
4. Direct reading electronic haemoglobinometer
5. Sahli’s method
, II. Measurement of O2 carrying capacity of Hb:
Measurement of O2 carrying capacity of Hb cannot be used for mass screening but is used
in referral or research laboratories only.
III. Measurement of iron content of Hb:
Measurement of iron content of Hb is used only for research purpose.
IV. Specific gravity method:
It is a very rapid method and is useful for screening blood donors for anaemia in blood
donation program. Normal specific gravity of blood ranges from 1.048-1.066.
Other methods like carboxy Hb and alkali haematin methods are not used these days.
Normal values of Hb
➢ Men: 15.5±2.5 g/dl
➢ Women: 14±2.5 g/dl
➢ Infants: 16.5±3 g/dl
Some of the commonly used methods are discussed below;
Cyanmethaemoglobin method:
This is the best method for Hb estimation and it has been recommended by International
Committee for Standarization in Haematoloy (ICSH).
Principle: Blood is diluted in a solution called Drabkin’s fluid containing potassium ferricyanide
and potassium cyanide. The oxy, carboxy, and metHb are all converted into cyanmetHb and there
is development of pink colour. The intensity of pink colour can be measured on photoelectric
colorimeter at 540 nm and this is compared with that of a standard cyanmethaemoglobin
solution.
Reagents: Drabkin’s fluid can be prepared as under;
• Haemoglobin (Hb) is the main component of red blood cells and is a conjugated protein.
• A molecule of Hb contains two pairs of polypeptide chains α2β2 and four haem groups
each having an atom of ferrous ion.
• The oxygen carrying capacity of Hb when fully saturated is 1.34 ml/g.
• Approximately 34% of the RBCs by weight is Hb.
• Iron content of Hb is 0.347gm/100 g.
• The main function of Hb is to transport oxygen from lungs to the tissues.
• There are various forms of Hb as under:
i. Oxyhaemoglobin (HbO2)
ii. Carboxy haemoglobin (COHb)
iii. Sulfhaemoglobin (SulfHb)
iv. Methaemoglobin (MetHb)
• The measurement of concentration of Hb in the blood is known as haemoglobinometry.
Types of blood sample used for estimation of Hb are as follows:
a. Capillary blood from finger prick
b. Intravenous sample: It should be well anticoagulated, preferably in EDTA. Liquid
anticoagulants should not be used at all because they dilute and decrease Hb
concentration.
Methods for estimation of haemoglobin
Various methods used for the estimation of Hb are divided into four groups as:
I. Colorimetric method:
Colorimetric method is based on colorimetric measurement of the intensity of colour
developed on addition of some substances to the blood. Colorimetric methods include
the following;
1. Cyanmethaemoglobin method
2. Oxyhaemoglobin method
3. Electronic counter method
4. Direct reading electronic haemoglobinometer
5. Sahli’s method
, II. Measurement of O2 carrying capacity of Hb:
Measurement of O2 carrying capacity of Hb cannot be used for mass screening but is used
in referral or research laboratories only.
III. Measurement of iron content of Hb:
Measurement of iron content of Hb is used only for research purpose.
IV. Specific gravity method:
It is a very rapid method and is useful for screening blood donors for anaemia in blood
donation program. Normal specific gravity of blood ranges from 1.048-1.066.
Other methods like carboxy Hb and alkali haematin methods are not used these days.
Normal values of Hb
➢ Men: 15.5±2.5 g/dl
➢ Women: 14±2.5 g/dl
➢ Infants: 16.5±3 g/dl
Some of the commonly used methods are discussed below;
Cyanmethaemoglobin method:
This is the best method for Hb estimation and it has been recommended by International
Committee for Standarization in Haematoloy (ICSH).
Principle: Blood is diluted in a solution called Drabkin’s fluid containing potassium ferricyanide
and potassium cyanide. The oxy, carboxy, and metHb are all converted into cyanmetHb and there
is development of pink colour. The intensity of pink colour can be measured on photoelectric
colorimeter at 540 nm and this is compared with that of a standard cyanmethaemoglobin
solution.
Reagents: Drabkin’s fluid can be prepared as under;
