HAEMOGLOBIN ESTIMATION
AIM : Estimation of haemoglobin level in a given sample solution.
Estimation of Hb level is called Haemoglobinometry.
Hb measurement can be done by two methods :
I. Direct
II. Indirect
INDIRECT METHODS:
Indirect methods of Hb measurement are called as Calorimetric methods
because Hb in RBCs in a given sample of blood is converted into a derivative of Hb and
then colour of that derivative is matched against standard colour either visually or by
photoelectric colourimetry.
Indirect methods are:
1. Sahli’s Method
2. Cyanmet Hb Method
3. Alkaline haematin Method
4. Oxyhaemoglobin Method
5. Haldane Method
6. Talliquist Method.
DIRECT METHODS:
In this, we measure oxygen carrying capacity of blood or do
iron estimation or by doing photospectrometry we estimate Hb
concentration.
DIRECT METHODS:
1. Oxygen carrying capacity: Hb forms aloose combination with oxygen.
Amount of oxygen that blood can carry is called oxygen carrying capacity
of blood. 1 gm of Hb can carry 1.34 ml of oxygen. Thus by measuring
amount of oxygen in given sample of blood,we can estimate blood Hb
conc.
2. Iron estimation of blood: by knowing iron content in a given sample of
blood, we can estimate Hb concentration.
SAHLI’s METHOD: this is an indirect method.
The readings are obtained in gm%.
APPARATUS REQUIRED: pricking needle, spirit swab, Sahli’s
haemoglobinometer, N/10 HCl, distilled water.
Sahli’s haemoglobinometer set:
It consists of:
A. Comparator: it is a rectangular plastic box with a slot in the middle
which accommodate calliberated Hb tube. Non-fading standardized
golden brown glass rods are fitted on each side of glass slot for
matching the colour. An opaque white glass is fitted at the back to
provide uniform illumination
B. Haemoglobin tube: also called Sahli-Adam’s tube. It is graduated in
gm% (from 2-24 gm%) yellow marking and on other side is
percentage (10-140%) red marking. 14.5 gm% corresponds to 100%.
C. Haemoglobin pipette: it has got a stem with a capillary space,
rubber tube and mouth piece. It has no bulb. There is only 1 mark
indicated on stem which is 20mm3.
PROCEDURE :
1. Clean the apparatus thoroughly.
2. With the help of dropper, put N/10 HCl into the haemoglobin
tube upto the lowest mark 20% or 2gm%.
3. Prick the finger with all aseptic precautions. Discard Ist drop of
blood.
4. Allow a large drop to form at the puncture site and suck blood
into the pipette upto 20mm3 mark.
5. Wipe the tip of the pipette so that no blood left sticking to the
outside of the pipette. Immediately blow this blood from the
pipette into the Hb tube containing N/10 HCl by dipping the tip
of pipette into HCl. Rinse the pipette 2-3 times by sucking and
expelling acid solution in Hb tube.
6. Mix the contents gently by shaking the tube. Put the tube back
into the haemoglobinometer and wait for 8-10 minutes.
During this time, cells rupture liberating Hb into acid solution.
Acid reacts with Hb to form acid hematin which is brown in
colour and is a stable compound.
7. After 10 minutes, take out the tube from Haemoglobinometer. Add a few
drops of distilled water and stir the contents gently, continue to add distilled
water drop by drop stiring the contents each time till the colour of solution
exactly matches with the standard.
8. Note the lower meniscus reading. Result is expressed gently in gm/100 ml of
blood.
PRECAUTIONS:
1. Apparatus should be thoroughly cleaned
2. Wipe off first drop of blood. Donot squeeze the finger.
3. Take care to avoid entry of air bubbles in the pipette.
4. There should be no blood sticking to the tip of the pipette.
5. Quickly transfer blood from pipette to Hb tube to prevent clotting of blood
inside the pipette.
6. Never take the stirrer out of the Hb tube otherwise some amount of acid
hematin gets removed. While colour matching, the stirrer should be held
above the level of solution.
7. Take the reading by holding the haemoglobinometer against natural light
in such a way that graduations donot interfere in matching of colour.
8. During colour matching, 3 readings should be taken
Normal Values of Haemoglobin :
1. Males: 14.5gm/dln(13.5 -18gm/dl)
2. Females : 12.5gm/dl ( 11.5-16gm/dl)
Merits and Demerits of Sahli’s Method:
MERITS :
1. This method is easy to perform and convenient.
2. Cost is very little as compared to other method.
3. It is a quick method.
4. Apparatus is portable and easy to handle.
5. No electric supply and technical knowledge is required.
DEMERITS:
1. The method requires visual comparison. So, subjective error
can occur.
2. With time, the colour of tinted glass rod may fade.
3. Acid hematin is not a true solution. Some precipitation may
occur.
4. This method does not detect all forms of Hb.
VARIATIONS:
A. If amount of Hb is less than normal, this condition is called Anaemia and
causes are:
i. Technical error
ii. Blood loss or trauma
iii. Deficiency of iron, vitamin B12 or folic acid
iv. Hookworm infestation causing iron deficiency anaemia.
B. If amount of Hb is more than normal, the condition is called polycythemia
and the causes are:
More erythropoiesis at high altitude.
