Determination of hemoblobin by acid hematin method by Prof. Dr. Kachiwal

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 https://www.facebook.com/ABKachiwal/ https://www.youtube.com/user/kachiwal2003 Email: [email protected] [email protected] Watsup# 03003058466

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

13. THANK YOU https://www.facebook.com/ABKachiwal/ https://www.youtube.com/user/kachiwal2003 Email: [email protected] [email protected] Watsup # 03003058466