# Haemoglobin Electrophoresis Interpretation

> Haemoglobin electrophoresis for NEET-PG Pathology: HbA2 cut-offs in beta-thalassaemia trait, HbF patterns, sickle and HbD migration at alkaline and acid pH, and HPLC.

- Canonical URL: https://prepelephant.com/topics/neet-pg/pathology/haemoglobin-electrophoresis-interpretation
- Exam / course: NEET-PG · Subject: Pathology
- Publisher: PrepElephant (https://prepelephant.com) — Prepared and reviewed by the PrepElephant Academic Review Team
- First published: 2026-10-02
- Last updated: 2026-10-02
- How to cite: "Haemoglobin Electrophoresis Interpretation", PrepElephant, https://prepelephant.com/topics/neet-pg/pathology/haemoglobin-electrophoresis-interpretation

## Direct answer

Cellulose acetate at alkaline pH 8.6 separates haemoglobins by net charge, and reading the lanes is a two-step discipline: name the bands by their position, then quantify the fractions against normal adult values (HbA 95-98 per cent, HbA2 1.5-3.5 per cent, HbF under 1 per cent). A raised HbA2 above 3.5 per cent with microcytosis defines beta-thalassaemia trait; HbF of 30-90 per cent with little or no HbA marks beta-thalassaemia major or intermedia; an HbS band with more HbA than S indicates sickle trait, while S dominant with absent A means disease or co-inherited thalassaemia. The critical limitation is that HbS, HbD and HbG co-migrate at alkaline pH — separation requires citrate agar electrophoresis at acid pH 6.2 or confirmation by a sickle solubility test — and HbE, common in eastern India, sits with HbA2 and HbC at the alkaline position.

## What you must remember

- **Normal fractions:** HbA 95-98 per cent, HbA2 1.5-3.5 per cent, HbF below 1 per cent in adults; HbF is physiologically high at birth (60-90 per cent) and falls over the first six months.
- **Beta-thalassaemia trait:** HbA2 above 3.5 per cent (typically 4-8 per cent) with a high-normal or slightly raised HbF; coexisting iron deficiency or delta-beta thalassaemia can lower HbA2 back toward normal and mask the trait.
- **Beta-thalassaemia major:** HbF 30-98 per cent with absent or minimal HbA; transfusion before sampling falsely restores HbA, so electrophoresis belongs before the first transfusion whenever possible.
- **Sickle quantitation:** sickle trait shows HbA exceeding HbS (about 55-65 versus 35-45 per cent); sickle disease shows HbS dominant with no HbA; HbF modulates severity through gamma-chain persistence.
- **Migration traps:** HbS, D and G travel together at alkaline pH; HbC, E and A2 travel together; acid pH electrophoresis on citrate agar separates S from D and C from E — the resolution step every exam probes.
- **Solubility test:** HbS polymerises in deoxygenated high-phosphate buffer, giving a turbid positive test, positive also in trait; it does not quantify.
- **Modern quantitation:** high-performance liquid chromatography and capillary electrophoresis measure fractions precisely and are the standard reporting platforms in Indian newborn-screening and antenatal thalassaemia programmes.

## Interpreting a microcytosis panel

A 24-year-old Bengaluru woman with a haemoglobin of 10.5 g/dL and mean corpuscular volume of 64 fL has a Mentzer index (MCV divided by haemoglobin) under 13, favouring thalassaemia trait over iron deficiency; ferritin is normal. Cellulose acetate electrophoresis at alkaline pH shows HbA2 quantified at 5.2 per cent with a minor HbF band — beta-thalassaemia trait, and the genetic counsellor's next request is haemoglobin studies in her fiance, because two trait parents face a one-in-four risk of a major-affected child per pregnancy — the core logic of Indian antenatal screening programmes. Contrast the newborn from a tribal district of Chhattisgarh with a band at the S position on HPLC with no HbA: sickle cell disease in a neonate whose HbF fraction is still high, entering penicillin prophylaxis and vaccination schedules early. Same instrument, two publics: counselling before marriage in one case, protection in infancy in the other.

## Where candidates slip

The band at the S position is announced as sickle without the second step — at alkaline pH that band may be D-Punjab or G, and only acid pH electrophoresis or the solubility test discriminates; the exam rewards the two-platform discipline. Iron deficiency is forgotten as the great masker: it suppresses HbA2 and can hide beta-thalassaemia trait until iron is repleted and electrophoresis repeated. In the opposite direction, a mildly raised HbA2 is over-called trait — megaloblastic anaemia, hyperthyroidism, unstable haemoglobins and antiretroviral therapy also nudge HbA2 upward in some patients, so borderline values need repetition. And co-inherited conditions reshape the picture: delta-beta thalassaemia deletes HbA2 synthesis while raising HbF, and HbE beta-thalassaemia, the double heterozygote common in West Bengal and the north-east, produces a severe transfusion-dependent phenotype from two individually mild variants.

## Frequently asked questions

### What HbA2 level defines beta-thalassaemia trait?

Above 3.5 per cent (typically 4-8 per cent), on electrophoresis, HPLC or capillary electrophoresis, in a microcytic patient with normal iron stores.

### Which haemoglobins co-migrate with HbS at alkaline pH?

HbD and HbG; distinguishing them requires citrate agar electrophoresis at acid pH or a sickle solubility test.

### How do sickle trait and sickle disease differ electrophoretically?

Trait shows both HbA (majority) and HbS (35-45 per cent); disease shows HbS dominant with absent HbA, HbF variable.

### What pattern does beta-thalassaemia major produce?

HbF of 30-98 per cent with little or no HbA and raised HbA2, best sampled before transfusion.

### Why can iron deficiency mask beta-thalassaemia trait?

Iron deficiency suppresses HbA2 synthesis, pulling the percentage back below the 3.5 per cent diagnostic cut-off until iron is repleted.

### Which compound heterozygosity is common in eastern India?

Haemoglobin E beta-thalassaemia, combining two mild variants into a severe, often transfusion-dependent phenotype prevalent in West Bengal, Assam and the north-east.