HEMOGLOBIN ELECTROPHORESIS

HEMOGLOBIN ELECTROPHORESIS

Electrophoresis is the movement of charged particles in an electric field. The speed of movement depends on the electrical charge on each particle and hydrogen ion concentration (pH) of the medium. At an alkaline pH of 8.4 to 8.6, 

Hemoglobin is negatively charged and migrates towards the anode. Due to structural variations in their molecules, different 

haemoglobins possess different electrical charges and therefore, separate during electrophoresis. 

Presence of abnormal hemoglobin can be detected by this method. Various support media such as paper, agar gel or cellulose acetate can be used for hemoglobin electrophoresis. 

Requirements

1. Electrophoresis chamber with power pack 

2. Cellulose acetate medium 

3. TRIS buffer, pH 8.4 

• TRIS EDTA 0.68 g 
• Boric acid 3.2 g 
• Distilled water1 Litre 

4. Ponceau stain (0.5%)

• Ponceau S 0.5gm
• Trichloroacetic acid 5.0gm
• Distilled water 100 ml 

5. Destaining and clearing agents

• 5 % acetic acid 
• Methyl alcohol
• 20 % acetic acid in absolute methyl alcohol 

6. Normal saline 

7. Abnormal haemoglobin control specimens.

Specimen EDTA anticoagulated blood 

Technique

Step 1: Preparation of haemolysate 

(1) Centrifuge the anticoagulated blood at 2500 rpm for five minutes.

(ii) Remove the plasma and wash the packed cells with large volumes of saline three times.

(iii) After the final washing, lyse the red cells by adding equal volume of distilled water, onequarter volume of toluene and one drop of 3 % potassium cyanide. Mix by inversion and centrifuge to remove the cell debris.

(iv) Transfer the haemolysate to a clean tube. 

Step II: Electrophoresis 

(i) Pour the buffer into the electropheresis chamber, soak the wicks and position them. Pre-soak the cellulose acetate plate for 20-30 minutes in the buffer. Remove the excess buffer by keeping the plate between absorbent papers.

(ii) Using an applicator, apply 0.5 to 0.6 ml of the specimen approximately 3 cm from the cathode. Also apply at least two abnormal controls on each plate.

(iii) Place the plate in the electrophoresis chamber. Place a microscope slide over it.

(iv) Run the electrophoresis at 450 volts for 20 minutes.

(v)Remove the cellulose acetate plate and stain with Ponceau S for three minutes.

(vi) Wash in three changes of 5% acetic acid.

(vii) Fix in absolute methyl alcohol for five minutes.

(viii) Clear in 20 % acetic acid in absolute methyl alcohol for 10 minutes.

(ix) Dry in oven at 65°C for 10 minutes.

(x) Scan the cellulose acetate plate with a scanning densitometer. 

Interpretation Compare the relative mobility of abnormal hemoglobin’s in the control samples with those of the test sample both visually and with scanning densitometer. Identify the abnormal hemoglobin’s present in the test sample.