Hemoglobin electrophoresis

Principle

• Haemoglobin variants differ in amino acid composition and therefore in net charge; in an electric field they migrate at different rates.
• Cellulose acetate at alkaline pH (8.4–8.6): Hb is negatively charged and moves towards the anode.
• Citrate agar (or acid agarose) at pH 6.0–6.2 separates variants that co-migrate at alkaline pH.

Specimen

• EDTA blood; washed red cells lysed to prepare a haemolysate.

Procedure

• Apply haemolysate and controls (e.g. A, F, S, C) to the medium near the cathode end.
• Run at constant voltage for the stated time in Tris-EDTA-borate buffer (alkaline) or citrate buffer (acid).
• Stain (e.g. Ponceau S) and compare bands with the control mixture; quantify by densitometry if required.

Results

• Alkaline, from origin (cathode side) towards anode: A2 = C = E = O-Arab; then S = D = G; then F; then A; H and Bart's fastest.
• Acid agar, cathode to anode: C, then S, then A with D, G, E and O near the origin, and F moving furthest towards the anode.
• Typical adult: HbA about 95–98%, HbA2 about 2–3.3%, HbF under 1%. Local ranges apply.

Interpretation

• HbA2 above about 3.5% supports beta-thalassaemia trait.
• AS trait: HbA greater than HbS. SS: mainly HbS with variable HbF and no HbA.
• High HbF: beta-thalassaemia major, HPFH, sickle cell disease, infancy.

Quality control and pitfalls

• HbA2 cannot be quantified accurately by alkaline electrophoresis because C and E co-migrate; use HPLC or capillary electrophoresis.
• Iron deficiency lowers HbA2 and can mask beta-thalassaemia trait.
• Transfusion within about 3 months invalidates results.
• Old samples show extra bands (e.g. methaemoglobin).

Clinical use

• Diagnosis of sickle cell disorders, HbC, HbE, HbD and beta-thalassaemia; antenatal and newborn screening.