What this test measures
High-performance liquid chromatography (HPLC) separates and quantifies the various haemoglobin fractions: HbA (normal adult), HbA2 (raised in beta-thalassemia trait), HbF (raised in beta-thalassemia major, sickle cell), HbS (sickle), HbC, HbD, HbE, and other variants. The chromatogram retention time identifies the variant and the percentage quantifies its contribution.
Why it matters
India has very high carrier rates of haemoglobinopathies in specific communities — beta-thalassemia trait ~4% nationally (15–20% in Sindhis, Punjabis, Gujaratis, some Bengali communities), sickle cell trait/disease in tribal Central India (Madhya Pradesh, Chhattisgarh, Maharashtra, Odisha) and parts of Kerala (10-30%), HbE in NE India (~50% in some Mongoloid tribes), HbD in Punjab. National Sickle Cell Mission (launched 2023) targets premarital, antenatal, and newborn screening. HPLC is the workhorse confirmatory test. Used for: premarital counselling, antenatal screening (couple screening), neonatal screening in high-burden states, evaluation of microcytic hypochromic anaemia not explained by iron deficiency, family screening of probands.
How to prepare
No fasting required. Disclose any recent blood transfusion (within 3-4 months may alter HbA2 and HbF percentages — test before transfusion or 3 months after). Note ethnicity / community / parents' regions of origin. Pregnancy and iron deficiency can lower HbA2 slightly — interpret with caution.
Markers & reference ranges
Reference ranges below are typical adult values. Your lab's reported range may differ slightly based on the assay platform and patient demographics — always read your report against the range printed on it.
| Marker | Normal range | If low | If high |
|---|---|---|---|
| HbA (%)[1] | 95 – 98 | Reduced HbA proportion suggests presence of variants displacing normal adult Hb. | Slightly raised — non-specific. |
| HbA2 (%) | 2.5 – 3.5 | < 2.5 — alpha-thalassemia (rare, needs DNA confirmation), iron deficiency, sideroblastic anaemia. | 3.5 – 4.5 — borderline beta-thalassemia trait. > 4.5 — beta-thalassemia trait confirmed (heterozygous). > 7 — possible HbA2 variant (Hb Lepore, delta-beta thal) or interpretation issue; confirm with DNA analysis. |
| HbF (%) | < 1 (adults); higher in newborns / children < 2 years | Low HbF in adults — normal. | Adult HbF > 1% — varies: 5-15% in beta-thalassemia trait, 30-90% in beta-thalassemia major, 5-30% in hereditary persistence of fetal Hb (HPFH), 10-50% in sickle cell disease. |
| HbS (%) | 0 (absence) | 0% — no sickle gene. | 20-45% with HbA dominant — sickle cell trait (AS, carrier). 50-90% with low HbA — sickle cell disease (SS or compound heterozygote SC, S-beta-thal). |
Common HPLC patterns
| Pattern | HbA | HbA2 | HbF | HbS | Diagnosis |
|---|---|---|---|---|---|
| Normal | 95-98 | 2.5-3.5 | <1 | 0 | Normal AA |
| Beta-thal trait | 92-95 | >3.5 | 1-5 | 0 | Heterozygous β-thal |
| Beta-thal major | 0-5 | Variable | 30-90 | 0 | Homozygous β-thal |
| Sickle cell trait | 55-60 | 2-3 | <1 | 40-45 | Heterozygous HbS (AS) |
| Sickle cell disease | 0-20 | 2-3 | 5-15 | 70-95 | Homozygous HbS (SS) |
| HbE trait | 60-70 | 2-3 | <1 | 0 (HbE 25-30%) | Heterozygous HbE |
| HbE/β-thal | Low | Variable | Raised | 0 (HbE 60-90%) | Compound heterozygous |
Frequently asked questions
Why is this test important in India?
India has ~40 million beta-thalassemia carriers and ~30 million sickle cell carriers concentrated in specific communities. Premarital and antenatal screening can prevent thousands of severely affected pregnancies — both diseases are autosomal recessive.
What if both parents are carriers?
Each pregnancy has 25% chance of severely affected child (thalassemia major or sickle disease), 50% carrier, 25% normal. Genetic counselling and prenatal diagnosis (chorionic villus sampling at 11–13 weeks) are options.
I have microcytic anaemia but my iron is normal — could it be thalassemia trait?
Very likely if you're from a high-carrier community (Sindhi, Gujarati, Punjabi, Bengali). Mentzer index (MCV/RBC count) < 13 favours thalassemia over iron deficiency. HPLC confirms.
How does pregnancy affect the result?
Pregnancy and iron deficiency can lower HbA2 slightly, making mild beta-thalassemia trait harder to detect. If clinical suspicion is high, repeat after pregnancy / iron repletion or do DNA analysis.
Will sickle cell affect my pregnancy?
Sickle cell trait (AS) is generally benign in pregnancy with extra hydration and avoiding hypoxia. Sickle cell disease (SS) carries higher pregnancy risk — maternal mortality, fetal growth restriction, preterm labour — needs multidisciplinary care.
Is this the same as Hb electrophoresis?
Functionally similar — both separate Hb variants. HPLC is more sensitive and quantitative; classical electrophoresis (cellulose acetate, then citrate agar) is older and still used as a confirmatory test for unusual HPLC peaks.
Related Autoimmune / Rheumatology tests
Tests commonly ordered alongside HEMOGLOBIN VARIANT ANALYSIS - HPLC, or that help interpret an unexpected result.
Sources & references
- ICMR — Haemoglobinopathies in India · accessed 2026-05-30T00:00:00.000Z
- WHO — Sickle Cell · accessed 2026-05-30T00:00:00.000Z
- BSH — Haemoglobinopathy Screening · accessed 2026-05-30T00:00:00.000Z
- Indian Society of Haematology and Blood Transfusion · accessed 2026-05-30T00:00:00.000Z
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