Determination of hemoglobin by acid hematin method is performed during online classes of 1st Prof.D.V.M. student of Sindh Agriculture University Tandojam.

1. DETERMINATION OF PACKED
CELL VOLUME (PCV)
BY
DR. ALLAH BUX KACHIWAL
PROFESSOR
DEPARTMENT OF VETERINARY PHYSIOLOGY AND BIOCHEMISTRY
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abkachiwal@sau.edu.com
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2. HEMOGLOBIN
 Introduction:
 Haemoglobin is the red pigment contained in erythrocytes, responsible for
transporting oxygen & carbondioxide.
 It consists of 4 Heme molecule + 4 globin chains. (iron-containing molecules
& protein chains)
 Its synthesis starts during red blood cells maturation particularly at
polychromatic erythroblast and continue untill reticulocyte stages.
 The main Hb type of normal adults is HbA, and little percentage of HbA2.

3. METHODS OF ESTIMATION OF HB
 Haemoglobin concentration (Hb) of a solution can be estimated by three
methods:
 1. Iron content method.
 2. Oxygen content method.
 3. Colorimetric methods

4. 1. IRON CONTENT METHOD
 By measuring the iron content of Hb.
 Every 100 g of Hb 0.347 g of iron.
 Disadvantages: Not suitable for routine work.

5. 2. OXYGEN CONTENT METHOD
 By measuring the oxygen content of Hb (Hb binding capacity)
 Every 1 g of Hb 1.34 ml of oxygen.
 Disadvantages: Not suitable for routine work (it is hardly practical).

6. 3. COLORIMETRIC METHODS-
 By measuring its color. –
 There are many colorimetric methods:
 i. Acid haematin method.
 ii. Alkaline haematin method.
 iii. Oxyhaemoglobin method.
 iv. Cyanomethaemoglobin method.
(The standardized method)

7. SAHLI’S ACID HEMATIN METHOD
 Principle –
 • Blood is mixed with an acid solution so that Hb is converted to brown
colored acid hematin
 • Diluted with water till brown colour matches that of brown glass standard •
Hb value is read directly from the scale

8. SAHLI’S ACID HEMATIN METHOD
 • Equipments-
 • Sahli hemoglobinometer
 • Sahli pipette(marked at 20 microlite or 0.02 ml)
 • Stirrer
 • Dropping pipette
 • Reagents
 • N/10 hydrochloric acid
 • Distilled water

9. PROCEDURE
 1. Fill the graduated Sahli tube to 20 mark with 0.1 N HCL.
 2. Add 0.02 ml of blood by immersing the nozzle of the pipette in the bottom
of the graduated tube, and gently blow the blood.
 3. Using the provided glass rod (mixer), mix the blood.
 4. Incubate for 5 – 10 min.
 5. Add D.W with pasteur pipette gradually untill the color of the diluted blood
in the test tube exactly matches the solid glass standard.
 6. Result is then read on Sahli scale. (100% = 14 g).

10.  Advantages
 • Easy to perform
 • Quick
 • Inexpensive
 • Can be used as a bedside procedure
 • Does not require technical expertise
SAHLI’S ACID HEMATIN METHOD

11. Determination of haemoglobin by Acid
Hematin Method

12. Matching of color and determination
of percentage of Hb

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Email: kachiwal2003@gmail.com
abkachiwal@sau.edu.com
Watsup# 03003058466