Haemoglobin is the main constituent of RBC. It carries out the important function of transportation of O2 from lungs to various parts of body and it also transports back CO2 from body to lungs. Haemoglobin consists of two components – Haem and globin.
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Haem is combination of iron and protoporphyrin and Globin is the protein, formed of amino acid chain.
Blood Specimen:
Capillary blood can be used directly. If veins blood is used, EDTA or heparin or double oxalate anticoagulants are used. There are various methods for estimation of Hb:
Principle:
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Hb is converted to acid haematin by the action of Hydrochloric Acid (HCl). The acid haematin solution is further diluted until its colour matches exactly to that of permanent standard comparable tube. The Hb is read directly from the graduated tube.
Procedure:
Fill the Hb cylinder up to the lowest mark with 0.1 normal HC1 solution. Add 20 µ liter (0.02 ml) blood with the help of Sahlis pipette. Make sure that there is no air bubble inside the pipette. Mix the blood with HC1, which is already placed in the cylinder.
Take care that there is no blood left to the sides of the cylinder. Rinse the pipette twice in the blood solution. Allow it to react for about 10 min. till the solution become dark brown in colour. Acid haematin is produced in cylinder after combination of Hb and acid.
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Dilute the solution by adding drop by drop of distill water, until perfect match is obtained with standard, comparable tube. Read the Hb concentration directly from the level of diluted solution. The reading may be in percentage (%) or gms/lit.
Colourimetric Method of Hb:
It is also known as cyanomethaemoglobin method.
Principle:
A small quantity of blood is taken and allowed to react with potassium cyanide and potassium fericynide (Drabkin’s solution). The chemical reaction gives product of stable colour. This product is called as cyanomethaemoglobin. The intensity of colour is directly proportional to Hb conc.
Equipment:
Colourimeter with green filter (540 nm.) Sahlis pipette, Test tubes, etc.
Reagent:
1. Drabkins solution.
2. Cyanomethaemoglobin – Standard solution
Composition:
i. Potassium cyanide – 50 mg.
ii. Potassium fericyanide – 200 mg.
iii. Distill water – 1000 ml.
Store in dark bottle at room temperature. Standard solution with Hb content of 5 gm, 10 gm, and 15 gm is recommended.
Procedure:
Take two test tubes and label it as ‘B’ (Blank) and T (Test solution) add 5 ml of Drabkins solution in each test tube. Avoid mouth pepetting as Drabkin solution is poison. Stopper the tube with rubber cap; add 0.02 ml of blood specimen into the tube marked with “I”.
The specimen is taken with the help of Sahlis pipette. Wipe off the tip of the pipette before adding blood into test tube. Mix the content of the tube and wait for 10 min with the help of blank and std, solution, find out absorption of test solution in colourimeter at 540 nm. Hb can be calculated as:
Hb conc. = Absorbance of test solution × Cone, of Std / Ab. of Std
Specific Gravity Method:
Principle:
When a drop of whole blood is dropped into a copper sulfate solution with a particular specific gravity, the density of drop is directly proportional to the amount of Hb in that drop. If the drop is denser than the specific gravity of solution, the drop is settled down to the bottom. If not, it will float on the top.
Procedure:
Copper sulfate solution of specific gravity 1.055 for male and 1.053 for female is taken in two test tubes. The specific gravity corresponds to minimum Hb level. For both the respective sexes 1.055 specific gravity corresponds to 13 gm/dl Hb and specific gravity 1.053 corresponds to 12 gm/dl of Hb.
A drop of blood is collected by skin puncture and taken into long necked pasture pipette and then immediately drops the blood into proper copper sulfate solution, observe for 15 to 20 sec.
If the person is normal, the drop of blood will settle down and if the person is anaemic, the drop will float on the top.
This test does not give the exact amount of Hb. This technique is usually applied in blood blanking for screening the door. It is quick and easy, accurate technique.
Gasometric Method:
In this method, Van silky apparatus is used for gasometric determination of Hb.
In this method blood is first of all saturated with oxygen. Then oxygen is taken off and collected, depending upon the amount of oxygen collected, the amount of Hb can be calculated.
For 1 gm of Hb -1.34 ml. of oxygen is present in blood.
Chemical Method:
In chemical method, Hb is found out by finding the amount of iron present in blood. The amount of iron is directly proportional to the amount of Hb present in blood.
Clinical Importance:
1. Decrease in Hb concentration below normal value, is sign of anaemia.
2. The Hb concentration is lower in adult female than in adult male.
3. Hb values are low during pregnancy due to haemodilution. Children also have low Hb.
4. An increase in Hb concentration can occur due to haemoconcentration, like loss of body fluid, in case of diarrhea, vomiting.
5. In Case of reduced oxygen supply, like heart disease, ploycythemia, Hb is low.
Decrease or increase in concentration of Hb should be reported, as it is a sign of disease.