Estimation of Haemoglobin Content

Aim: To determine the haemoglobin (Hb) content of own blood by Sahli’s haemoglobinometer. 

Requirements:  0.1 N HCl, distilled water, sterile disposable needle (26G), cotton swab, 70% alcohol or any other suitable marketed antiseptic and Sahli’s Haemoglobinometer.

Sahli's Haemoglobinometer 
Fig.1: Sahli’s Haemoglobinometer 

Sahli’s Haemoglobinometer consists of:

  • Comparator: It is a rectangular plastic box with a slot in middle to accommodate the calibrated haemoglobin tube. The golden-brown tinted glass rods are fitted on each side of the slot for matching the colour. Opaque white glass is fitted behind the glass rod to provide uniform illumination.
  • Haemoglobin tube: It is a calibrated glass tube with marking of g Hb% i.e. g of Hb in 100 ml of blood (2-24 g Hb%) in yellow colour on one side and %Hb (20-140%) in red colour on other side. The brush is provided to clean the tube.
  • Haemoglobin pipette: It is an 8-10 inches long glass capillary pipette with only a single calibration mark of 20 µl or 0.02 ml.
  • Stirrer: It is a thin glass rod with a flattened end that is used for stirring and mixing the blood, acid and water.
  • Pasteur pipette: It is an 8-10 inches long glass tube with a long thin nozzle with a rubber teat.


Haemoglobin (Hb) is a red pigment present in the RBCs. It consists of ‘Haem’ as an ‘Iron fraction’ and ‘globin’ as a ‘protein fraction’. The globin contains four polypeptide chains (two α and two β chains) each consisting of one haeme pigment. Each haeme bears one iron molecule which can combine with one oxygen (O2) molecule. Hb estimation is a part of a routine blood test. The levels of Hb are significant to establish conditions like anaemia, leukaemia, chronic infections, certain cancers, etc. 

The haemoglobinometry is a measurement of the Hb concentration of blood. Sahli’s method is based on characteristics of Hb like the presence of the known amount of iron in each gram of Hb and the ability of Hb to reflect specific wavelengths of light. When the whole blood is added to dilute HCl, it causes haemolysis releasing the Hb and forming acid haematin. This gives golden brown colour after dilution with distilled water. The intensity of colour produced depends upon the concentration of acid haematin which in turn depends upon the concentration of Hb. The colour of the solution after dilution with water is matched against golden brown tinted glass rods by direct vision. The readings are obtained in g% (gram per cent). 

This method is simple, quick and accurate, does not require costly and sophisticated apparatus and is hence one of the most widely accepted methods. 


  1. Fill the Hb tube up to the mark of 20% with 0.1 N HCl. 
  2. Get a finger prick under aseptic conditions and fill the pipette up to the mark of 20 µl. 
  3. Once the pipette is filled, immediately immerse the tip of the pipette to the bottom of the acid solution and expel the blood gently. Rinse the pipette 3-4 times by drawing up and blowing out the 0.1 N HCl solution. 
  4. Mix the blood with 0.1 N HCl solution with the help of the flat end of the stirrer. 
  5. Put the tube into the comparator and let it stand for 6-8 minutes (during this period formation of acid haematin takes place). 
  6. Dilute this acid haematin solution with distilled water till the colour matches that of standard tinted glass rods in the comparator. 
  7. Read the lower meniscus of the coloured solution and note the reading as Hb g%. 

Note: The average level and range of Hb are as follows:

  • Males: 14.5 g% (13.5-18.0 g%).
  • Females: 12.5 g% (11.5-16 g%).

Observation table: 

  • Name: 
  • Age: 
  • Gender: 
  • Date: 
Sr. No.ReadingHaemoglobin content (g%)

Result:  The haemoglobin content was found to be ‘x’ g%. 

Clinical Significance: 

Hb concentrationIndications/Factors responsible 
1. More than normalPolycythemia Increased viscosity of blood 
2. Less than normalAll types of anaemia
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