BBCCL-116 Human Physiology: Lab
EXPERIMENT 5
TO ESTIMATE THE
HAEMOGLOBIN CONTENT IN A
BLOOD SAMPLE
Structure
5.1 Introduction 5.4 Procedure
Expected Learning 5.5 Result and Observations
Outcomes
5.6 Precautions
5.2 Principle
5.7 Terminal Questions
5.3 Materials Required
5.1 INTRODUCTION
Haemoglobin (Hb) is the respiratory pigment which transports oxygen
throughout the body and gives red color to the blood. It consists of globin and
heme. In a healthy person, its amount remains constant and is found to be 14-
Note: Refer to section 5.7 18g/dL in males and 12-16g/dL in females. The 1 gm of Hb can hold and carry
unit 5 Respiratory upto 1.34 mL of oxygen. Decreased concentration of Hb below this normal
physiology, for more
range causes anemia, while increased level of Hb in blood causes
details of structure and
polycythemia. Therefore, determination of Hb level in blood is an important
function of haemoglobin
test for detecting anemia in human beings. Sahli’s haemoglobinometer is
commonly used for determination of Hb percentage in human blood.
Expected Learning Outcomes
After studying and performing this exercise, you should be able to:
state the principle of Sahli’s method;
enlist the components of haemoglobinometer;
perform the method to know haemoglobin content in your blood; and
calculate haemoglobin content in gram/dL;
30 discuss the normal value and interpretation of abnormal results.
Experiment 5 To Estimate the Amount of Hemoglobin in a Blood Sample
5.2 PRINCIPLE
Haemoglobin gets converted to the brown-coloured acid hematin in the
presence of HCl. The intensity of brown colour depends on the amount of
hemoglobin present in the blood sample. The acid hematin solution is diluted
until it’s colour exactly matches with the standard brown colour present in the
glass tube of the comparator box (Haemoglobinometer). The concentration of
Hb then can be read directly from this hemoglobinometer.
5.3 MATERIALS REQUIRED
1. Haemoglobinometer or Hemometer
2. Hb pipette
3. Test tube
4. Glass rod
5. Dropper
6. N/10 HCl
7. Distilled water
Haemoglobinometer: It is a set of equipment which contain a comparator, Hb comparator box with
hemoglobin tube, hemoglobin pipette and a stirrer (Fig.5.1). brown glass on either side
and tube.
Fig. 5.1 Schematic diagram of Hemoglobinometer.
The Comparator contains two sealed glass tubes carrying brown-coloured
standard for colour matching. Between the two tubes, there is a slot to
accommodate the graduated hemoglobin tube for blood sample.
Haemoglobin tube is graduated tube also called the Sahli-Adams tube. It is
marked on one side in gram percent (%) from 2 to 22 and on the other side in
percentage (%) from 10 to 140.
Hb pipette is graduated up to mm3and is used for sucking the blood. 31
BBCCL-116 Human Physiology: Lab
Stirrer is a thin glass rod used for stirring the solution.
Dropper is used for addition of distilled water/solution into the test tube.
5.4 PROCEDURE
1. Clean and dry haemoglobinometer tube and pipette.
2. Fill the hemoglobin tube with N/10 HCl upto the mark 2g% using
Using the web link, you can dropper.
watch a YouTube video
called "Estimation of 3. Prick the finger carefully following aseptic precautions and discard first
haemoglobin by Sahli's drop of blood.
method."
4. Press the finger for larger drop of blood and draw the blood in the pipette
https://www.youtube.com/wat upto 20 mm3 mark (0.02mL blood).
ch?v=-EDI-EN9MJU
5. Wipe the tip of the pipette to remove any excess blood.
6. Immediately transfer the blood from the pipette into the haemoglobin
tube containing N/10HCl. Blow the pipette two or three times into the
acid solution to empty the pipette. Mix the blood with the acid thoroughly
using the stirrer.
7. Leave the tube in comparator undisturbed for about 10 minutes to form
the acid hematin (brown in colour).
8. Now using dropper, dilute the solution by adding distilled water drop-by-
drop stirring it with a glass rod. After adding each drop of water, match
the colour of solution in the tube with that of the standard of the
comparator glass. While matching the colour, the glass rod must be
removed from the solution.
9. When the colour of the solution matches exactly with the colour of the
standard, record the reading directly by holding the haemoglobinometer
at eye level.
5.5 RESULT AND OBSERVATIONS
Express the concentration of haemoglobin as g% of blood (gram per 100 mL
blood). Analyze your result. Normal value of blood Hb is14-18 g/dL in males
and 12-16 g/dL in females.
WHO recommended reference values for anemia are:
Males <13 g/dL
Females <12 g/dL
Pregnant women <11 g/dL
5.6 PRECAUTIONS
1. Make sure that the blood sample is not coagulated.
2. Do not take large volume of N/10HCl.
32
Experiment 5 To Estimate the Amount of Hemoglobin in a Blood Sample
3. Wait for minimum 10 min after mixing blood with the HCl to develop acid
hematin.
4. Blood should be transferred immediately from the pipette to the tube
having HCl solution to prevent blood coagulation.
5. The colour of acid hematin should be checked immediately after addition
and mixing each drop of distilled water.
6. Keep the stirrer away while comparing the color of solution of tube glass
in the comparator box.
7. Always compare the color of the solution with the color of standard box
by holding hemoglobinometer at your eye level.
5.7 TERMINAL QUESTIONS
1. What is a haemoglobinometer, and how does it work?
2. What is the normal haemoglobin value in males and females?
3. What is the purpose of the HCl in Sahli's method?
33
