2. 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
3. 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.
4. 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.
5. 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.
6. Requirements
Sahli’s hemoglobinometer
N/10 Hcl
Distilled water
Two Dropper
Materials for sterile finger prick
7.
8.
9. 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. 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,
1st reading , when the colour is slightly darker than the
std……………..g/dl.
2nd reading, when, after adding a few drops of distilled
water the colour exactly matches with std ………….g/dl.
3rd reading, when, after adding some more drops, the
colour becomes a little lighter than the
std……………g/dl.
Hb=………………g/dl.
12. 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.
13. 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)
14. 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.
15. 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.
16. 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. 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.
II. 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.
19. Conditions that increase Hb Conc.
I. Physiology
High altitude (due to hypoxia)
New borns and infants
Excessive sweating (due to hemoconcentration)
II. 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.
20. 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?