Title: Haemoglobin Estimation by cyanmethaemoglobin method
is the major component that present in red blood cell. The main function of
haemoglobin is to carry out transportation of oxygen from the lungs to rest of
the body. And it also carried out transportation of carbon dioxide from the
body and back to lungs, carbon dioxide is to be exhaled out through the process
of respiration. Haemoglobin consists of four molecules of haem group and one
molecule of globin. In each haem group, there is present of an atom of iron
whereas globin is a pair of polypeptide chain formed by series of amino acid.
One haemoglobin capable to combine with four molecules of oxygen molecules.
state (Fe2+) of iron in haemoglobin undergo oxidation to ferric ions
(Fe3+), methaemoglobin is formed. Potassium ferricyanide acting as
the oxidising agent in this process. Then, methaemoglobin reacts with potassium
cyanide and form cyanmethaemoglobin. Cyanmethaemoglobin
is a compound with stable red colour, it can be measured colorimetrically. The
intensity of cyanmethaemoglobin is directly proportional to the amount of
haemoglobin in the sample.
Specimen: EDTA anticoagulated blood is
preferred. The specimens may be collected also with heparin, citrate or oxalate
Reagent: 1) Drabkin’s reagent
Sodium bicarbonate -1g
Potassium Ferricyanide – 200g
Potassium cyanide – 50mg
Distilled water – 1L
Note: Cyanide is poisonous
and must be handle with care. Drabkin’s solution is in pale yellow colour and
should be kept in brown bottle.
2) Methaemoglobin standard (20g)
Apparatus: Culture tube, spectrophotometer,
Two culture tubes are labelled
as BLANK and PATIENT respectively.
Both tubes are filled with 5 ml
of Cyanmethemoglobin reagent.
20µl of blood sample is
pipetted into the tube PATIENT. BLANK
tube is left only with cyanmethemoglobin.
The tube are allowed to stand
for 10 minutes.
Spectrophotometer is adjusted to zero
absorbance at 540 nm when using the tube BLANK.
cuvette with fluid from BLANK and PATIENT. Insert cuvette of BLANK reagent in
the spectrophotometry following by cuvette of PATIET. Absorbance values for both
are examined and recorded.
equation is used to determine unknown concentrations:
(g/dl) = Sample absorbance X
Standard Concentration (g/dL)
A 20 g/dl Standard had
an absorbance of 0.500, and the unknown absorbance is 0.395. The hemoglobin concentration of the unknown is:
0.395 X 20 = 14.4 g/dl
Adult male: 13.0 – 18.0 g/dl
Adult female: 11.5 – 16.5
For the experiment that we carried out, we
found out the absorbance value for 20g/dl of BLANK is 0.456 and absorbance
value of PATIENT is 0.454. Thus, the haemoglobin concentration is
Sample absorbance is
directly proportional to the concentration of haemoglobin. The haemoglobin is
the main component that make the red blood cell appear in red colour. As the
concentration of haemoglobin increase in the blood, the reddish colour of blood
will turns deeper and it will increase the absorbance of sample. The objective
of carrying out this experiment is 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