Hb electrophoresis and its types, principle , procedure

Trying to simplify the concept of hemoglobin electrophoresis for bachelor and diploma students.
Hopefully this slide helps.

1. Haemoglobin Electrophoresis Prepared by : Sumina Kainchi Sujan Kawan

2. Electrophoresis is defined as the process by which molecules are seperated on the basis of migration of charged particles under the influence of an electrical field. Factors electrophoresis depends on , Net charge of molecule Size and shape of particle Strength of electrical field Ionic strength of buffer Properties of supporting medium INTRODUCTION :

3. Supporting media for electrophoresis Cellulose acetate Agarose gel Polyacrylamide gel Starch Paper

4. Hb electrophoresis is the movement of hb proteins in a electric field at a fixed Ph. Hb electrophoresis uses an electrical current to seperate normal and abnormal types of Hb in the blood. Hb types have different electrical charges and move at different speeds. Hb Electrophoresis:

5. 1.Normal Hb : a. Hb A (95-98%) b. Hb A2 ( 2-3%) c. Hb F 2. Abnormal Hb: a. Hb S b. Hb C c. Hb E Types of Hb :

6. 1.Cellulose acetate at alkaline pH 2.Citrate agar at acidic pH Methods of Hb Electrophoresis

7. Cellulose acetate is highly compatible with alkaline pH (typically pH 8.0–9.0). In this pH range, hemoglobin molecules are more negatively charged due to the deprotonation of acidic amino acid residues (such as glutamic acid). Requirement : Haemolysate Tris-EDTA boric acid buffer(Ph 8.4) Whatman No.3 filter paper Cellulose acetate membrane Protein stain solution (0.5% ponceau S) Cellulose acetate at alkaline pH

8. In an alkaline pH (8.2-8.6) Hb is a negatively charged molecule and will migrate toward the anode (+). The various Hbs moves at different rates depending on their net negative charge, which in turn is controlled by the composition (amino acids) of the Hb molecule (globin chain). Identification of different haemoglobins is based on their relative positions on cellulose acetate strip. Principle of cellulose acetate

9. The hemolysate is prepared in order to destroy the red cell membrane and to free the Hb . Preparation of hemolysate from whole blood :

10. 1.0.50ml blood in a test tube 2.Add 5ml normal saline, mix and centriguge at 1500RPM for 5 minutes.(repeat for 3 times) 3.Add 0.15ml of distilled water,mix well. 4.Then haemolysate is ready. Procedure to prepare haemosylate

11. Set Up Electrophoresis : Add buffer and wet wicks in the tank. Soak Cellulose Acetate : Soak cellulose acetate in buffer for 5 minutes. Load Samples : Load 5 μl of each sample into wells, cover with coverslip to prevent evaporation. Run Electrophoresis: Apply samples to the cellulose acetate, place in the tank, and run at 350 V for 25 minutes. Staining : Stain with Ponceau S for 5 minutes, wash, blot, and dry. Procedure

12. Control Sample: Always run a control sample with known hemoglobins (like Hb A, Hb F, Hb S, and Hb C) alongside your test sample to compare results. Movement on the Strip: Hemoglobins move to different places on the electrophoresis strip based on their charge: Hb A moves the farest . Hb F (fetal hemoglobin) moves slower than Hb A. Hb S (sickle cell hemoglobin) moves slower than Hb A. Hb C has a movement between Hb A and Hb S. Interpretation

13. Diagnosis: By comparing the movement of the test sample to the control, you can identify normal or abnormal hemoglobins .

14. Citrate agar seperates Hb fractions that migrate together on cellulose acetate agar. In an acidic medium, hemoglobin molecules exhibit different migration patterns compared to alkaline conditions because the net charges of hemoglobins change due to protonation of their amino acid residues. At a lower pH, hemoglobins gain protons (H+ ions), which alters their net charge and electrophoretic mobility. Hemoglobins with more positively charged amino acid residues migrate slower than those with fewer positive charges. Citrate agar Electrophoresis

15. Citrate agar electrophoresis is used to confirm variant Hbs and further differentiate HbS from HbD and G and HbC from HbE . The procedure should not be used as a screening procedure because many abnormal Hbs migrate with HbA . However, this procedure is a method of choice when examining newborns(cord blood) and infants under 3 mnths for some abnormal Hbs such as S and C as it is able to detect quantities of Hb not easily seen by other techniques .

16. Thank you !!!