Determination of Haemoglobin is performed by First Prof. D.V.M. students and presented in class

2.  Sher Muhammad pirzada
(Group Leader)
 Shah Inayat
 Zahid hussain
 Shahjahan
 Zarab ali
 Zulfiqar ali
 Syed Imran
 Shahyan Ayoub
 Zeeshan
 Wajid
 Tehseen Raza
 Waleed Ahmed
 Sijawal
 Waniya Menal Siddique
(Group Leader
 Zarisht Rao
 Vanila
 Shubaiqa
 Siddiqa
 Taqadus
 Sheena
Group members

3. Submitted To
Dr. Allah Bux Kachiwal
Professor & Chairman
Department of Physiology
and Biochemistry

5. Learning Points:
• Introduction of Hemoglobin
• Normal ranges of Hemoglobin
• Determination of Hemoglobin by Acid
Hematin Method

6. Determination of Hemoglobin
by Acid Hematin Method

7. • Hemoglobin is the iron-containing oxygen
transporter metalloprotein in the red blood cells of
humans. It gives the blood its red color.
• Hemoglobin carries oxygen from the lungs to the
rest of the body ( i.e the tissues ) where it releases
the O2 to burn nutrients to provide energy to
power the functions of the body & collects the
resultant CO2 to bring it back to the lungs to be
releases to the atmosphere .
• Hemoglobin is the major constituent of the red
cell cytoplasm.
Hemoglobin

8. • Hemoglobin molecule is a tetramer consisting of
two pairs of similar polypeptide chains called
globin chains.
• To each of the four chains is attached heme
which is a complex of iron in ferrous form and
protoporphyrin. It gives red color to the blood.
• The major (96%) type of hemoglobin present in
adults is called HbA and it has 2 alpha globin
chains & 2 beta globin chains (α2β2)
Structure of Hemoglobin

9. Structure of
Hemoglobin

10. Male
Female
Neonate
14-18
12-16
13-20
The normal value of Hb varies according to
the age and sex of the individuals. The
normal ranges are:
Normal HB
levels

11. Age group Hemoglobin count
(gm/dl)
Normal Hemoglobin Range in New born
babies
13-20
Normal Hemoglobin Range in Children 11-13
Normal Hemoglobin Range in Adults
(Male)
14-18
Normal Hemoglobin Range in Adults
(Female)
12-16
Normal Hemoglobin Range in Elderly
(Male)
12.4-14.9
Normal Hemoglobin Range in Elderly
(Female)
11.7-13.8
Normal hemoglobin
values

12. Methods
Of HB
Estimation
Manual
Acid Hematin
Method by
(Sahili
Method)
Cyanmethemoglobi
n
Method
Automated
Coulter
Counter
Method of Hemoglobin
Estimation

13. • Sahli hemoglobinometer
• Sahli pipette(marked at 20 microlite or
0.02 ml)
• Stiirrer
• Dropping pipette
• Reagents
• Hydrochloric acid
• Distilled water
Sahli’s Acid Hematin Method
• Equipments:

14. • Sahli’s/acid Hematin Method Principle: Blood is
mixed with N/10 HCl resulting in the conversion of
Hb to acid Hematin which is brown in color.
• The solution is diluted till it’s color matches with the
brown colored glass of the comparator box.
• The concentration of Hb is read directly.
Sahli’s Acid Hematin Method
Principles:

15. .
1. Take a spirit swab. Thoroughly clean the fingertip and prick the pulp of the
finger with the disposable Lancet.
2. Discard the first drop of blood and allow the hemoglobin pipette to the full by
capillary action up to mark 20.
3. Place the tip of the pipette beneath the surface of HCI in the Sahli‘s tube and
gently blow out the blood in the tube.
4. Take Sahli’s hemoglobin tube and with the help of a dropper. Add 0.1 N (N/10)
HCI in the tube and place it in Sahli‘s hemoglobinometer
5. Mix the contents with the glass stirrer and allow it to stand for 10 minutes. The
RBCs hemolysis during this time. Hemoglobin releases and reacts with HCI and
gets convert into acid Hematin.
6. Then add distilled water drop by drop “me stirring the contents until the color
matches with the color of the standard in the comparator.
7. Remove the Sahli’s tube and hold it against white light or paper.
8. Read the upper meniscus on Sahli’s tube to measure the percentage of
hemoglobin and grams of hemoglobin per 100 ml of blood.
Sahli’s Acid Hematin Method
Procedure:

16. Video Demonstration
https://youtu.be/Ctph-w2kmf8

17. 1. The blood sample and acid should while taking
should be of the accurate and precise amount because
it can affect the results.
2. While Sucking blood, Don’t allow the air bubbles to
enter the pipette.
3. Always use the disposable lancet. Thoroughly clean
the puncture site with antiseptic.
4. Colour matching should be carry out during good
daylight.
5. Always take the absolute values, as %age values have
no significance.
Sahli’s Acid Hematin Method
Precaution:

18. We got the result of Waniya Menal
Sidddique’s hemoglobin
conentration which is 12gm/100
ml

19. 1. Method is very simple
2. Easy to perform
3. Method is not time-consuming.
4. Cheap and can be used for hemoglobin
estimation where automated hematology
analyses are not available.
5. Does not require technical expertise
Sahli’s Acid Hematin Method
Advantages: