Estimation of Hemoglobin by Sahli’s Method
BP107P Human Anatomy and Physiology Practical
To determine the haemoglobin content in 20µl of the blood sample.
Haemoglobin (Hb) is a red pigment present in the RBCs. It is the tetrapyrrole porphyrin ring, It consists of ‘Haem’ as an ‘Iron fraction’ and ‘globin’ as a ‘protein fraction’. The globin contains four polypeptide chains (two a and two B chains) each consisting of one haeme pigment. Each haeme bears one iron molecule which can combine with one oxygen (O₂) molecule. Hb estimation is a part of a routine blood tests. Each amino acid chain is attached to a heme moiety to form a single haemoglobin molecule. Each RBC contains about 280 million haemoglobin molecules and each haemoglobin can carry up four oxygen molecules.
A haemoglobin molecule consists of a protein called globin. It is composed of four polypeptide chains i.e. two alpha and two beta chains, plus four non-protein pigments called themes. One ring-like heme binds to each polypeptide chain. At the centre of the heme, the ring is an iron ion (Fe²+) that can combine reversibly with one oxygen molecule. One gm of haemoglobin carries 1.34 ml of oxygen. In addition to its key role in transporting oxygen and carbon dioxide, haemoglobin
The levels of Hb are significant to establish conditions like anaemia, leukaemia, chronic infections, certain cancers, etc.
Haemoglobin carries the O₂ from lung to tissue and the CO₂ from tissue to lungs and also plays a role in the regulation of blood flow and blood pressure.
Functions of Haemoglobin
i. It transports oxygen from the lungs to the tissues by forming oxyhaemoglobin. Oxygen combines reversibly to an iron ion of the heme ring of the haemoglobin molecule.
ii. It transports about 13% of the total carbon dioxide, waste products of metabolism. Carbon dioxide combines with amino acids in the globin part of haemoglobin.
iii. It acts as a buffer in maintaining blood pH.
iv. On breakdown it forms important bile pigments.
After the normal life span is over, the red blood cells are destroyed by the macrophages in the liver, spleen or red bone marrow into heme moiety (biliverdin and iron) and globin protein. The iron and globin are recycled to reuse and the biliverdin is converted into the bilirubin which is further converted into the stercobilin to be removed in the faeces.
The Colour of the faeces is yellow because of the presence of stercobilin.
Types of Haemoglobin (Hb)
i) Adult Haemoglobin:
(a) Hb A: It consists of two a₂ chains and two ß chains in globin protein, a₂ß₂ 97% of adult Hb is of this type.
(b) Hb A₂ (α₂62): Minor Hb A₂ present in adults, the concentration of Hb increases in some types of anaemia.
(c) Fetal haemoglobin:
Hb F (a₂Y2): Hb F accounts for 70-90% of haemoglobin at term, 25% in one month, and 5% in six months.Hb F concentration in adults increases in some types of anaemia, haemoglobinopathies, and some time in leukaemia.
Haemoglobin Bart’s (y4): Its concentration increases in fetal life in thalassemia.
ii) Embryonic haemoglobin:
Hb Gower 1 (22), Hb Gower 2 (₂2), and Hb Portland (0212).
A hemoprotein composed of globin and heme that gives red blood cells their characteristic colour; functions primarily to transport oxygen from the lungs to the body tissues. The red blood cells are broken down with hydrochloric acid to get the haemoglobin into a solution. The free haemoglobin is exposed for a while to form hemin crystals. The solution is diluted to compare with a standard colour.
Haemometer, Single mark pipette, Distilled water, Needle, Spirit, Cotton, HCl.
Time needed: 15 minutes
How to determine the haemoglobin content?
- Take Haemoglobin tube
Take 1/10 HCl in the Hb tube upto the lowest mark ‘2’.
- Pricking finger
Prick the finger with the needle
- Collection of blood sample
Collect 20µl of the blood sample with a single mark pipette.
- Formation of hemin crystals
Place the Hb tube on the working table for five minutes for the formation of hemin crystals.
- Dilution of sample
Place the Hb tube in the compater/hemometer and add drop by drop of distilled water into it until the colour of the solution in the Hb tube coincides with the glass plates of the compater.
- Observation and reading
If the colour coincides with the glass plates of the compater, observe the reading in the Hb tube. The percentage of Hb can be calculated from the reading
Hb content in grams X 100 / 14.5 NORMAL VALUES: Males = 14 to 18 grams
Females = 13 to 14 grams
Children = 10 to 13 grams
The haemoglobin content present in 20µl of blood sample is __________
Synopsis Questions and Answers
1. Method is very simple
2. Method is not time-consuming.
3. Cheap and can be used for haemoglobin estimation where automated haematology analyses are not available.
4. No technical expertise is needed to perform.
5. does not require costly and sophisticated apparatus and is hence one of the most widely accepted methods.
1. There is about 8% to 10% of error in the estimation of haemoglobin.
2. This method does not estimate all the haemoglobins (estimate only oxyhaemoglobin and reduced haemoglobin, but haemoglobin in blood is also present in other forms like Carboxyhemoglobin, methemoglobin, and sulfhemoglobin which are not converted to acid hematin so not detected.
3. The method is less accurate because the brown colour of acid haematin does not remain stable; 95% colour of acid hematin is formed in 10 mins it fades with time or fades almost immediately after it reaches its peak.
4. It is difficult to match perfectly with the glass comparator visual errors can be there.
5. Acid hematin solution is not stable and colour formation is slow, sometimes precipitation may occur.
6. Source of light will influence the visual comparison of colours.
7. Color of the brown glass standard fades with time so it is not reliable.
8. Individual variation in the matching of colour is seen.
1. Do not take the excess volume of N/10 HCI (not above the 20% mark) in the tube.
2. Hemoglobin pipette and Sahli’s tube should be clean and dry before use.
3. The pricking should be done boldly.
4. Do not squeeze the finger since there is a possibility of dilution of blood with other body fluids.
5. The first drop of blood should be discarded as it is mixed with tissue fluid.
6. Suck the blood exactly up to 20 cu mm (20 uf) mark in the pipette.
7. There should not be any air bubbles in the pipette with blood.
8. Wipe off the tip of the pipette before transferring blood from the pipette to the haemoglobinometer tube.
9. The blood should be immediately transferred to a tube containing HCI to prevent clotting in the pipette.
10. Wait for 8-10 minutes after adding the blood in acid.
11. Wait for a minimum of 10 minutes after mixing blood with HCI for complete conversion of haemoglobin to acid haematin.
12. The colour of the acid haematin should be checked frequently with the addition of each drop of water so distilled water to be added drop by drop to avoid over dilution.
13. The tube should be placed in the comparator in such a way that the graduations on it do not lie directly in front, which may interfere with the matching of colour
14. The matching of colour should be done by holding a haemoglobinometer at eye level against a natural source of light or electrical tube light (white light).
a. Sahli’s method
b. Dare’s method
c. Haden’s method
d. Haldane’s method
e. Wintrobe method
f. Copper sulphate method
g. Tallquist method
h. Gasometric method
I. Oxyhaemoglobin method
j. Cyanmethemoglobin method
k. Alkaline haematin method
l. Specific gravity method
m. Iron estimation method
n. Comparator method
It is a rapid method for estimation of the approximate level of Hb and it is used in large surveys.
In this method, a drop of blood is absorbed on an absorbent paper and the colour of this paper is matched with the standard.
Gasometric method of estimation of haemoglobin is done by using van Slyke apparatus,
It is the most accurate method. However, it is time-consuming and a complex procedure. Only used for research purposes and for standardization.
In this method, blood is first treated with potassium cyanide and potassium ferricyanide, which forms methemoglobin. Light absorbed by this is compared with a standard solution in the photoelectric calorimeter
Blood is mixed in a solution containing potassium cyanide and potassium ferricyanide. The potassium ferricyanide converts Hb to methemoglobin which is converted to cyanmethemoglobin (HiCN) by potassium cyanide. The absorbance of the solution is then measured in a spectrophotometer at a wavelength of 540nm. It can also be measured in a colourimeter using a yellow-green filter.
Alkali is used to form alkali hematin and the colour is matched to standard
This method only gives an estimate of haemoglobin and not a quantitative method. It is a quick and easy method done to screen blood donors for possible anaemia
A drop of blood is dropped into a copper sulphate solution of particular specific gravity. The drop falls rapidly if the specific gravity of blood is greater than the specific gravity of copper sulphate, otherwise, it may fall slowly or may float.
Blood with normal haemoglobin levels falls rapidly.
Blood with low haemoglobin concentration- falls slowly or floats.
It is based on the principle that 100 gm Hb contains 347 mg of iron. The value of iron is estimated in the blood which gives the value of Hb.
Male adult: 15.5 gm/100 ml (14-18 gm %)
Female adult: 14 gm/100 ml (12-15.5 gm%)
Newborn: 16.5 gm/100 ml
1. To determine (if less than the normal value of Hb in blood its an indication)
2. Screening for polycythemia (if more than normal value its an indication)
3. To determine the viscosity changes in the blood (higher values observed than the normal with an increase in viscosity of blood)
4. To assess response to a specific therapy in anaemia.
5. Estimation of red cell indices
6. Selection of blood donors.
Even if more amount of N/10 HCI is taken it would not make much difference when haemoglobin content is normal. But if a person has severe anaemia then the colour produced may be lighter than the standard and correct estimation then can not be done, if more quantity. of N/10 HCI is taken.
In Sahli’s method of estimation of haemoglobin exact quantity of blood (20 mm³) is taken. It is made to react with HCI to form acid haematin. For conversion of entire haemoglobin present in the blood to acid haematin dilute HCI up to 20% mark is required. If a lesser amount of HCI is taken, the entire haemoglobin may not be converted to acid haematin and wrong results would be obtained.
No, we can not use either HNO, or H,SO, because they are strong oxidizing agents and do not form coloured acid haematin with haemoglobin
No, tap water cannot be used for dilution as it causes turbidity and interferes with the colour of the solution. However, N/10 HCI can be used for dilution as it does not change the colour of the solution.
Haemoglobin concentration in a newborn baby is high. It may be as high as 20 g/100 ml of blood. (Due to more no. of active sites of red bone marrow compared to adults).
The normal range of haemoglobin concentration of cord blood is 16.5 ± 3 g/100 ml of blood in the full-term babies.
After several days, the haemoglobin level progressively falls up to 12 g ± 1g/100 ml at the 12th month of life. Then level rises to adult value by puberty. In males, the Hb value is 1.5 g/100 ml higher than in females. Mean values of haemoglobin then remain constant until the age of about 70 years. Then the levels progressively fall by approximately 0.5 g/100 ml per decade.
As compared to males haemoglobin concentration is lesser in females due to -1) Loss of blood during menstruation. 2) In males, testosterone hormone stimulates RBC production leading to high RBC count and high haemoglobin concentration.
From haemoglobin values following indices are calculated
1) MCH = Mean Corpuscular Haemoglobin. Its normal value is 27 to 32 picograms 2) MCHC = Mean corpuscular haemoglobin concentration. The normal value is 32 to 38%. 3) CI= Colour Index. Its normal value is 0.8 to 1.2. Indices help in diagnosing the type of anaemia.
Human Anatomy and Physiology Practical Syllabus
- Study of a compound microscope.
- Microscopic study of epithelial and connective tissue
- Microscopic study of muscular and nervous tissue
- Identification of axial bones
- Identification of appendicular bones
- Introduction to hemocytometry.
- Enumeration of white blood cell (WBC) count
- Enumeration of total red blood corpuscles (RBC) count
- Determination of the bleeding time
- Determination of clotting time
- Estimation of haemoglobin content
- Determination of blood group.
- Determination of erythrocyte sedimentation rate (ESR).
- Determination of heart rate and pulse rate.
- Recording of blood pressure.
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