Title: Haemoglobin Estimation by cyanmethaemoglobin method Introduction:Haemoglobinis the major component that present in red blood cell. The main function ofhaemoglobin is to carry out transportation of oxygen from the lungs to rest ofthe body. And it also carried out transportation of carbon dioxide from thebody and back to lungs, carbon dioxide is to be exhaled out through the processof respiration.
Haemoglobin consists of four molecules of haem group and onemolecule of globin. In each haem group, there is present of an atom of ironwhereas globin is a pair of polypeptide chain formed by series of amino acid.One haemoglobin capable to combine with four molecules of oxygen molecules.
Methaemoglobin(Fe3+) Haemoglobin(Fe2+) Principle: The ferrousstate (Fe2+) of iron in haemoglobin undergo oxidation to ferric ions(Fe3+), methaemoglobin is formed. Potassium ferricyanide acting asthe oxidising agent in this process. Then, methaemoglobin reacts with potassiumcyanide and form cyanmethaemoglobin. Cyanmethaemoglobinis a compound with stable red colour, it can be measured colorimetrically. Theintensity of cyanmethaemoglobin is directly proportional to the amount ofhaemoglobin in the sample. Materials:Specimen: EDTA anticoagulated blood ispreferred. The specimens may be collected also with heparin, citrate or oxalateas anticoagulants. Reagent: 1) Drabkin’s reagent Sodium bicarbonate -1g Potassium Ferricyanide – 200g Potassium cyanide – 50mg Distilled water – 1LNote: Cyanide is poisonousand must be handle with care.
Drabkin’s solution is in pale yellow colour andshould be kept in brown bottle. 2) Methaemoglobin standard (20g) Apparatus: Culture tube, spectrophotometer,cuvette, micropipette Methods:1. Two culture tubes are labelledas BLANK and PATIENT respectively.2. Both tubes are filled with 5 mlof Cyanmethemoglobin reagent. 3. 20µl of blood sample ispipetted into the tube PATIENT.
BLANKtube is left only with cyanmethemoglobin.4. The tube are allowed to standfor 10 minutes.5. Spectrophotometer is adjusted to zeroabsorbance at 540 nm when using the tube BLANK.
6. Filled thecuvette with fluid from BLANK and PATIENT. Insert cuvette of BLANK reagent inthe spectrophotometry following by cuvette of PATIET.
Absorbance values for bothare examined and recorded. Results/Observation:Calculation: The followingequation is used to determine unknown concentrations: Patient sample(g/dl) = Sample absorbance X Standard Concentration (g/dL) Standard absorbance Example: A 20 g/dl Standard hadan 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 0.550 Expected Values:Adult male: 13.0 – 18.
0 g/dl Adult female: 11.5 – 16.5g/dl For the experiment that we carried out, wefound out the absorbance value for 20g/dl of BLANK is 0.456 and absorbancevalue of PATIENT is 0.454. Thus, the haemoglobin concentration is Discussion: Sample absorbance isdirectly proportional to the concentration of haemoglobin.
The haemoglobin isthe main component that make the red blood cell appear in red colour. As theconcentration of haemoglobin increase in the blood, the reddish colour of bloodwill turns deeper and it will increase the absorbance of sample. The objectiveof carrying out this experiment is to detect anemia and its severity and tomonitor anemic patient under the treatment. It is also used to check the Hb level ofpotential donor’s blood, prior of donation