Estimation of Hemoglobin (hb) by Pandian M, Tutor, Dept of Physiology, DYPMCKOP,MH.

What is Hemoglobin?
Practical
Requirements
How to prepare N/10 Hcl
Procedure
Observation & Result
Oxygen carrying capacity
Iron Content
Advantage & Disadvantage
Normal Levels
Questions

1. M.Pandian
Dept of Physiology

2. SLO
 What is Hemoglobin?
Practical
Requirements
How to prepare N/10 Hcl
Procedure
Observation & Result
Oxygen carrying capacity
Iron Content
Advantage & Disadvantage
Normal Levels
Questions

3. What is Hemoglobin?
Hemoglobin (abbreviated Hb) is a red substance made of
iron and protein
Carries Oxygen to the cells
Carries Carbon dioxide away from the cells

4. Each molecule of hemoglobin contains four groups of
heme.
Heme is the iron component of hemoglobin.
Each group of heme can carry one molecule of oxygen.

5. A Hemoglobin molecule contains four
globin polypeptide chains composed of
amino acids.
Amino acids are a group of chemical
substances that form proteins.
Each hemoglobin molecule consists of
about 3.8% heme and 96.2% globin.

6. Practical
Aim
To determine the Hb content of blood (sahli’s method
its indirect method.)
Principle
The amount of Hb can be estimated by the
conversion of the known volume of blood into
acid-haematin by the addition of dilute N/10
Hcl and subsequent calorimetric comparison
with suitable standard.

7. Requirements
Sahli’s hemoglobinometer
N/10 Hcl
Distilled water
Two Dropper
Materials for sterile finger prick

10. How to prepare N/10 Hcl
Normality (N)
The normality of a solution is the number of gram equivalents in
1 litre of water.

A 1 N solution of Hcl contains 1 + 35 = 36 g of Hcl in water made
to 1 liter.
Diluting this solution 10 times will give N/10 Hcl.

11. Procedure

12. Observation & Result
Compare your colour matching and record the
observations in your work book.
Take average of 3 readings as shown below, and
report your results,
 1
st
reading , when the colour is slightly darker than
the std……………..g/dl.
2
nd
reading, when, after adding a few drops of distilled
water the colour exactly matches with std ………….g/dl.
3
rd
reading, when, after adding some more drops, the
colour becomes a little lighter than the std……………
g/dl.
 Hb=………………g/dl.

13. Oxygen carrying capacity
Knowing your Hb Conc. and that 1.0 g of Hb can
carry 1.34 ml 0f O2, calculate its O2 carrying
capacity as ……………..ml O2/dl.
Normal values :- Males – 21mL/dl ; Female –
18mL/dl
Iron Content
1g of Hb contains 3.35 mg iron, the amt of Hb can
be calculated.

14. Advantage
Sahli’s method (indirect method)is easy to perform and
convenient.
The cost is minimal.
It is not very time consuming (maximum fifteen minutes)

15. Disadvantage of Sahli’s method
The colour of the std may not always be reliable,
especially with old apparatus.
Sahli’s acid haematin method does not estimate
all the Hb. It estimate only oxyHb and reduced
Hb, but not the carboxyHb, methemoglobin and
sulfphhemoglobin.
Finally, the acid haematin is not true solution.
Some degree of precipitation may be present at
times, which may interfere with colour matching.

16. Normal Levels
12 to 16 (14 ± 2) grams per deciliter (g/dl)of blood in women
13.5 to 18 (16 ± 2) g/dl of blood in men
New born, 16 – 22 g/dl. It decreases to 9 – 14 g/dl by abt two months of
age. By 10yrs of age, the normal Hb Conc. Will be 12 - 14 g/dl.
There may be a slight decrease in Hb level after 50 yrs of age.

17. Other Methods
Visual method
Dare’s method
Haden’s method
Wintrobe’s method
Haldane’s method
Tallquist’s method
Gastrometric method
Spectrophotometric (these methods are rapid and give accurate result)
Oxyhemoglobin method
Cyanmethemoglobin method
Automated hemoglobinometry
Non-Automated hemoglobinometry
Alkaline- hematin method
Specific gravity method
Comparator method

18. Precautions

19. Conditions that decrease Hb Conc.
I.Physiology
Pregnancy (due to hemodilution)
Children have lower values than adults
Women have lower values than men because the total
RBC count is less.
For male its due to testostreone stimulates
erythropoiesis in males.
I.Pathology
Different types of anemia
Relative decrease in Hb Conc. Occurs in different
pathological conditions that produce hemodilution,
for e.g excess ADH secretion as seen in pituitary
tumors.

20. Conditions that increase Hb Conc.
I.Physiology
High altitude (due to hypoxia)
New borns and infants
Excessive sweating (due to hemoconcentration)
I.Pathology
Conditions that produce hemoconcentration (due to
loss of body fluid) for example, severe diarrhea,
vomiting.
Conditions that produce hypoxia (e.g. congenital
heart disease, emphysema.
Polycythemia vera.

21. Questions
1.What is N/10 Hcl and how will you prepare it?
2.Can strong acids ( such as Nitric, Sulphuric,& Hcl) or
alkalins be used in place of decinormal Hcl.
3.Why is the Hb level high in the new born?
4.What would happen if Hb present freely in the
plasma instead of in the red Cells?
5.Why should ten mins be allowed before diluting the
solution of blood & N/10 Hcl?

22. Test Time
1) Hemoglobin (abbreviated______) is a red substance made of iron and
protein.
Hb
2) Hemoglobin __________ Oxygen to the cells.
Carries.
3) Carries __________away from the cells
Carbon dioxide

23. 4) _______is the iron component of hemoglobin
Heme
5) Each group of heme can carry _____ molecule of oxygen
One
6) Normal hemoglobin is 12 to 16 grams per deciliter (g/dl) in
women T/F
True

24. References
Text book of Medical
Physiology
 Guyton & Hall
Hutchinson Clinical Methods
Practical Physiology Manual
A.K. Jain, C.L. Ghai, G.K. Pal
Net source for pictures

25. Thank You