Haemoglobin Electrophoresis and HPLC Interpretation
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Direct answer
Haemoglobin electrophoresis and HPLC separate normal and abnormal haemoglobins, and they are the standard laboratory tests for thalassaemia and sickle cell disorders. Interpretation rests on HbA2 and HbF levels, the band pattern and any HbS or HbE, read with the red cell indices. An HbA2 above about 3.5 per cent points to beta thalassaemia trait, whereas a normal HbA2 with microcytosis raises suspicion of alpha thalassaemia, which needs genotyping.
What you must remember
- Normal adult pattern: HbA is about 95 to 98 per cent, HbA2 about 2 to 3.5 per cent, and HbF is usually under 2 per cent.
- HbA2 threshold: a value above about 3.5 per cent is the key marker of beta thalassaemia trait, often reaching 4 to 7 per cent.
- HbE co-migration: HbE runs with HbA2 on alkaline electrophoresis and elutes in the HbA2 window on common HPLC systems, falsely raising the apparent HbA2, so a large A2-window peak is confirmed by acid-pH electrophoresis, capillary electrophoresis or DNA analysis.
- HbH disease: a fast-moving HbH band and red cell inclusions on supravital staining mark the deletional alpha thalassaemia form.
- Iron deficiency: low iron lowers HbA2 and can mask beta thalassaemia trait, so iron status is checked before interpretation.
- Sickle cell: the sickle solubility test is positive in both trait and disease, so HbS must be quantified to separate them.
- Hb Barts: gamma-4 haemoglobin seen at birth and on newborn screening suggests alpha thalassaemia when it persists or is high.
Reading the HbA2 and HbF pattern
A 25-year-old woman from eastern India has microcytosis with an MCV of 68 fL and a normal serum ferritin. Her HPLC shows an HbA2 of 5.2 per cent and no HbS or HbE. The pattern must be read against the red cell indices and the iron status.
| Pattern | HbA2 | HbF | Key remark |
|---|---|---|---|
| Normal adult | About 2 to 3.5% | Under 2% | HbA about 95 to 98% |
| Beta thalassaemia trait | Above 3.5%, often 4 to 7% | Normal or mildly raised | Microcytosis with normal ferritin |
| Alpha thalassaemia trait | Normal | Normal | Genotyping needed for confirmation |
| HbH disease | Normal or low | Variable | HbH band and inclusions on staining |
| Beta thalassaemia major | Raised | Predominant | Little or no HbA |
| HbS trait (AS) | Normal | Normal | HbS below half of total haemoglobin |
| HbSS disease | Normal or mildly raised | Raised | No HbA, HbS predominates |
| HbE trait | Falsely raised (HbE in A2 window) | Normal | HbE co-migrates with HbA2 |
Her raised HbA2 with a normal ferritin points to beta thalassaemia trait rather than iron deficiency. Her partner should be tested, since the carrier state matters for counselling.
Where the exam tries to catch you
Electrophoresis questions usually hinge on the HbA2 value and on what can falsely change it.
- Trap: diagnosing beta thalassaemia trait from microcytosis alone Correct: an HbA2 above 3.5 per cent confirms the trait, and iron deficiency must be excluded because it lowers HbA2.
- Trap: reading a raised HbA2 in a patient with HbE on alkaline electrophoresis Correct: HbE co-migrates with HbA2 and often elutes in the A2 window on HPLC, so a large A2-window peak needs confirmation by acid-pH or capillary electrophoresis or DNA analysis.
- Trap: assuming a normal HbA2 excludes thalassaemia Correct: alpha thalassaemia often has a normal HbA2, so genotyping or HbH testing is needed.
- Trap: reporting a positive sickle solubility test as a diagnosis Correct: the test is positive in trait and disease alike, so haemoglobin separation is required.
Frequently asked questions
What HbA2 level indicates beta thalassaemia trait?
An HbA2 above about 3.5 per cent, often between 4 and 7 per cent, indicates beta thalassaemia trait. The value is interpreted only after excluding iron deficiency, which lowers HbA2 and can mask the trait. Coexisting alpha thalassaemia can also modify the result in some patients.
Why can haemoglobin E falsely raise the HbA2 reading?
HbE migrates at the same position as HbA2 on alkaline electrophoresis and elutes in the HbA2 window on common HPLC systems, so the HbE band is read as HbA2. This gives a falsely raised HbA2 (often 25 to 35 per cent) that could be mistaken for beta thalassaemia trait. Acid-pH or capillary electrophoresis, or DNA analysis, confirms HbE.
How is HbH disease identified in the laboratory?
HbH disease is identified by a fast-moving HbH band on alkaline electrophoresis and by red cell inclusions on a supravital stain such as brilliant cresyl blue. Its HbA2 is typically normal or low.
Why must iron status be checked before reading an HbA2 value?
Iron deficiency lowers HbA2 and can hide beta thalassaemia trait, producing a falsely normal result. Serum ferritin is therefore measured, and iron deficiency is treated before the study is repeated. A raised HbA2 that persists after iron repletion strongly suggests the trait.
What does a positive sickle solubility test actually show?
A positive sickle solubility test shows that HbS is present, but it cannot tell sickle cell trait from sickle cell disease. Haemoglobin electrophoresis or HPLC quantifies HbS and separates the two conditions.