To estimate the haemoglobin content in a sample of your own blood by Sahli’s method.
Haemoglobin present in a sample of blood is converted into acid hematin by addition of N/l 0HClto the blood and its haemoglobin content is determined by matching the solution against non fading glass having a standard colour.
APPARATUS:Sahli’s haemoglobinometer (haemometer), distilled water, rectified spirit, cotton, lancet.
SAHLI’S HAEMOGLOBINOMETER consists of:
1. Comparator box: with colour standards on either side with a space for keeping the diluting tube in the middle.
The standard is non fading yellowish brown tinted glass, the colour of which is that of acid haematin obtained by
treating blood containing 14.5gm of haemoglobin per decilitre, with 0.1 NHCL and diluting it 100 times
2. Special diluting tube: which is graduated in gm/dl scale on one side and percentage scale on the other side(1 4.5gm/dI corresponding to 100%).
3. Haemoglobin pipette: which is a capillary pipette with a 20 mm3 marking
4. Glass stirrer
5. A bottle containing Nil OHCL
PROCEDURE:Take NI 10 HCI upto the lowest mark in the diluting tube. Keep the diluting tube in the space provided in the box. Sterilize the fingertip using rectified spirit. Make a quick prick to get moderately large drop of blood. Suck the blood in the haemoglobin pipette upto the 20mm3 mark without any air bubble. Wipe off any blood sticking to the tip and sides of the pipette using cotton. Transfer the blood immediately into the acid taken inthe diluting tube. Rinse the pipette two or three times with the acid and transfer into the diluting tube. Mix and keep it undisturbed for 10 minutes, so that haemoglobin gets converted to acid haematin. After 10 minutes, dilute the contents by adding distilled water drop by drop and mixing the contents after each drop with the stirrer, till the colour matches with the colour of the standard. Then take the reading both in gram scale and percentage scale by noting the lower meniscus.
1. There should not be any air bubble or blood clot in the column in the pipette.
2. Graduations on the diluting tube should not interfere with colour matching.
3. The glass rod should be lifted up before colour matching and reading.
4. Wipe off the excess blood sticking to the sides and tip of the pipette.
5. Transfer the contents immediately into the diluting tube and note the time.
6. Take the reading without any delay because on keeping the colour will deepen.
1 Individual variations in colour matching.
2. Colour of the standard fades with time.
3. Incomplete conversion of haemoglobin to acid haematin (sulf haemoglobin, met haemoglobin and carboxy haemoglobin will not form acid haematin).
4. Presence of proteins and lipids in plasma will interfere with colour matching. Other Methods:
1. Colorimetric methods:
a. Direct matching
i) Taliquist paper scale method
ii) Dane method
b. Indirect matching:
Alkali haematin method: This is a better method for estimation of haemoglobin. Even carboxyhaemoglobin, methaemoglobin etc. are converted into alkali haematin.
2. Gasometric method:
This is based on the amount of gas bound by haemoglobin and then released.
1. Oxygen carrying capacity method
2. CO saturation method.
3. Photometric methods:
Visual comparison or colour matching with the naked eye is subject to individual variations, which can be avoided by photo electric colorimetry. Here haemoglobin may be estimated as oxyhaemoglobin by the use of 0.1% sodium carbonate solution, acid haematin by the use of N/10 HCI.
4. Method depending on the iron content of blood
5. Physical methods
1. Making use of specific gravity
2. Spectroscopic method
Males: 14-18gm! lOOmI
Females: 12-16gm! lOOmI
Newborns: 18-23gm/i OOml
At high altitude haemoglobin content is increased. Hypoxia stimulates erythropoiesiswhich in turn increases haemoglobin content. Haemoglobin content is increased after exercise.
Pathological variations: Haemoglobin content is increased in polycythemia and decreased in all types of anaemias.
Functions of haemoglobin:
1. Transport of oxygen
2. Transport of CO2
3. It acts as a buffer
4. Bilirubin formation
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