Subtitle: A case-based guide to haemoglobin electrophoresis and cation-exchange HPLC: how the methods work, the HPLC windows, interpreting HbA2 and HbF, the patterns of beta thalassaemia trait, HbE, HbS, HbC, HbD, Hb Bart’s and HbH disease, pitfalls such as iron deficiency and co-eluting variants, and 10 case-based MCQs.
Author: PathologyMCQ Editorial Board
Category: Haematology
Last Updated: October 2026
Read Time: 8 minutes
At-a-Glance
- HbA2 above 3.5% with microcytosis = beta thalassaemia trait; iron deficiency can mask it.
- A2 window above 10% = a co-eluting variant (HbE, Lepore, D-Iran), not true HbA2.
- Sickle trait: HbA greater than HbS (35–40%). Disease: no HbA. S/beta-plus: HbA less than HbS.
- Alkaline electrophoresis cannot separate S from D/G or C from E/O; acid agar can.
- Hb Bart’s at birth grades alpha thalassaemia; HbH disease shows a fast peak and golf-ball inclusions.
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The Methods
How haemoglobin variants are separated
| Method | Principle | Strengths | Limits |
|---|---|---|---|
| Cellulose acetate electrophoresis, pH 8.6 | Charge at alkaline pH; cathode to anode: C/E/O/A2, S/D/G/Lepore, F, A, H/Bart’s | Cheap, visual, good for screening | S = D = G and C = E = O; poor HbA2 quantitation |
| Citrate agar electrophoresis, pH 6.2 | Charge plus interaction with agar; cathode to anode: C, S, A (with D, G, E, O), F | Separates S from D/G and C from E/O | Not quantitative |
| Cation-exchange HPLC (for example Bio-Rad Variant, D-10) | Elution by increasing ionic strength; each haemoglobin has a retention time window | Quantifies HbA2 and HbF; detects most variants; automated | Co-elution (HbE with A2, HbD/G near S); HbH and Bart’s may be under-estimated |
| Capillary zone electrophoresis | Charge in a capillary at alkaline pH, UV detection | Quantitative; separates HbE from HbA2 and HbS from HbD | Different zones from HPLC; needs its own experience |
| Confirmatory tests | Sickle solubility, isoelectric focusing, mass spectrometry, globin gene PCR/sequencing | Identify the variant and the genotype | Not first line |
HPLC Windows and Retention Times
Typical Bio-Rad Variant II beta-thalassaemia short programme windows
| Window | Retention time (min) | What elutes there |
|---|---|---|
| F | about 1.0–1.2 | HbF (and Hb Bart’s, HbH earlier, often under-estimated) |
| P2, P3 | 1.3–1.9 | Glycated HbA1c, carbamylated and degraded Hb |
| A0 | about 2.0–2.6 | HbA |
| A2 | about 3.3–3.9 | HbA2, HbE, HbLepore, HbD-Iran, some HbC-Harlem |
| D | about 3.9–4.3 | HbD-Punjab, HbG-Philadelphia, HbQ-India |
| S | about 4.3–4.7 | HbS and some HbG variants; confirm with a solubility test |
| C | about 4.9–5.3 | HbC, HbO-Arab, HbC-Harlem |
Windows vary with the analyser and programme, so always read the chromatogram, not only the percentage: look for abnormal peaks, retention time shifts and a total area within limits.
Normal Values and What Changes Them
- HbA 96–98%, HbA2 2.2–3.5%, HbF below 1% in adults (higher in infancy, pregnancy and HPFH).
- HbA2 raised: beta thalassaemia trait (3.5–7%), megaloblastic anaemia, HIV antiretrovirals, hyperthyroidism, and spuriously by HbE, Lepore or D-Iran co-elution (above 10%).
- HbA2 lowered: iron deficiency, alpha thalassaemia (slightly), delta thalassaemia, HbH disease, recent transfusion.
- HbF raised: beta thalassaemia major and intermedia, delta-beta thalassaemia, HPFH, sickle cell disease, stress erythropoiesis, hydroxyurea, JMML, pregnancy.
Patterns to Recognise
Haemoglobinopathy patterns on HPLC
| Condition | HbA | HbA2 | HbF | Variant | Red cell indices |
|---|---|---|---|---|---|
| Beta thalassaemia trait | Normal | 3.5–7% | Normal or up to 5% | None | MCV low, RBC count high, RDW normal |
| Beta thalassaemia major (untransfused) | Absent (beta-zero) or reduced | Variable | Up to 95% | None | Marked microcytosis, anaemia |
| Alpha thalassaemia trait | Normal | Normal or low | Normal | None (Bart’s 3–8% at birth) | MCV low; HPLC normal in adults |
| HbH disease | Reduced | Low | Normal | HbH 5–30%, fast peak | MCV low, haemolysis, golf-ball inclusions |
| Sickle cell trait | 55–65% | Normal | Normal | HbS 35–40% | Normal |
| Sickle cell anaemia (SS) | Absent | Normal or slightly raised | 2–20% | HbS over 80% | Normal MCV unless alpha thal |
| S/beta-plus thalassaemia | 5–30% (less than S) | Raised | Raised | HbS 60–90% | MCV low |
| HbSC disease | Absent | Normal | Low | HbS and HbC about 50% each | Mild microcytosis, target cells |
| HbE trait | 65–75% | in A2 window 25–30% | Normal | HbE | Mild microcytosis |
| HbE/beta thalassaemia | Absent or low | in A2 window | Raised | HbE | Thalassaemia intermedia or major picture |
| HPFH (deletional, heterozygous) | Normal | Low-normal | 15–35%, pancellular | None | Normal |
| Delta-beta thalassaemia trait | Normal | Low-normal | 5–20%, heterocellular | None | MCV low |
Pitfalls
- Recent transfusion adds donor HbA and dilutes everything else; wait three months or test pre-transfusion.
- Iron deficiency lowers HbA2 and can push beta thalassaemia trait into the normal range; correct iron first.
- Newborns have mostly HbF; interpret with neonatal cut-offs and report in the FAS/FS/FSC/FAC style.
- HbA2 cannot be quantified reliably in the presence of HbS on some systems; glycated HbS forms a shoulder.
- Co-elution: always confirm an unexpected peak with a second method (acid agar, capillary electrophoresis, solubility or DNA).
- A normal HPLC does not exclude alpha thalassaemia; use red cell indices, Hb Bart’s at birth and alpha-globin PCR.
Test Yourself: 10 Case-Based MCQs
Haemoglobin electrophoresis and HPLC: 10 case-based MCQs
Thalassaemia traits, HbS, HbE, HbD, Hb Bart’s, HPFH and the pitfalls, with instant explanations.
Score: 0 / 10
More practice: the question banks →
Exam Pearls
- Microcytosis + high red cell count + HbA2 above 3.5% = beta thalassaemia trait.
- A2 above 10% is a variant, usually HbE.
- Trait: A more than S. Disease: no A. S/beta-plus: A less than S.
- Acid agar separates what alkaline cannot: S from D/G, C from E/O.
- Bart’s at birth: 1–2% silent, 3–8% trait, 20–40% HbH.
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References
- Bain BJ. Haemoglobinopathy Diagnosis. 3rd ed. Wiley-Blackwell; 2020.
- Bain BJ, Bates I, Laffan MA, eds. Dacie and Lewis Practical Haematology. 12th ed. Elsevier; 2017.
- Ryan K, et al. Significant haemoglobinopathies: guidelines for screening and diagnosis. Br J Haematol. 2010;149:35–49.
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