Hematology & Laboratory Medicine & Family Medicine
Hemoglobin electrophoresis
Hemoglobin electrophoresis is a laboratory technique for separating, identifying and quantifying the different hemoglobin fractions present in circulating erythrocytes, by exploiting the differences in electrical charge of globin chains according to their amino acid composition. Human hemoglobin is a tetrameric protein composed of two pairs of globin chains (two alpha chains and two non-alpha chains), each associated with an iron-carrying heme group. In healthy adults, three hemoglobin fractions normally coexist: hemoglobin A (HbA - α₂β₂ - 96 to 98 % of the total), hemoglobin A2 (HbA2 - α₂δ₂ - 2 to 3,5 %) and fetal hemoglobin (HbF - α₂γ₂ - <1 % in adults - up to 70 to 90 % in neonates before the switch to globin synthesis at 6 months). Hemoglobinopathies - mutations or deletions of globin genes - constitute the most widespread group of monogenic diseases in the world (over 300 million carriers). They fall into two main categories: structural hemoglobins (substitution of an amino acid on the globin chain - hemoglobin S in sickle cell disease, hemoglobins C, D, E) and thalassemias (quantitative decrease in the synthesis of one or more normal globin chains - alpha-thalassemia and beta-thalassemia). Hemoglobin electrophoresis - or its modern replacement, high-performance liquid chromatography (HPLC) - is the gold standard for diagnosing these diseases, and is an essential tool for neonatal screening, prenatal genetic counseling and follow-up of patients with known hemoglobinopathies. In Quebec, universal newborn screening for hemoglobinopathies using HPLC (Programme québécois de dépistage néonatal - PQDN) has been in force since 2009 in certain regions, and is gradually being extended.
Indications, technique, and normal values
- Clinical indications for hemoglobin electrophoresis: hemolytic anemia of undetermined etiology (especially if microcytic hypochromic without iron deficiency - thalassemia); suspicion of sickle cell disease (child or adult from Sub-Saharan Africa, the Caribbean, North Africa, the Middle East, India, or the Mediterranean presenting with vaso-occlusive crises, acute chest syndrome, palpable spleen, or splenic sequestration episodes); pregnancy workup (woman from an endemic area or known carrier partner); prenatal genetic counseling (both parents carrying a hemoglobinopathy trait - 25% % risk of homozygous form); family screening after diagnosis of a hemoglobinopathy; microcytic hypochromic anemia with normal ferritin (minor thalassemia - elevated HbA2); unexplained polycythemia (hemoglobin with high oxygen affinity); universal neonatal screening (PQDN Quebec)
- Available techniques: HPLC (high-performance liquid chromatography): the current reference technique - separation of hemoglobin fractions by affinity on a cation-exchange column - precise quantification of all fractions (HbA, HbA2, HbF, HbS, HbC, HbD, HbE, etc.) - results within a few minutes.) - results in 10 to 15 minutes - used for neonatal screening in Quebec (LSPQ); capillary electrophoresis (CE): separation by electric charge in a buffer-filled capillary - excellent precision and reproducibility - complementary to HPLC for rare hemoglobins; cellulose acetate gel electrophoresis (alkaline pH) + citrate agar electrophoresis (acid pH): classic techniques still used in reference laboratories - the combination of the two pH values enables distinction between hemoglobins that co-migrate at a single pH (eg. HbS and HbD co-migrate at alkaline pH but separate at acid pH); falciformation test (sickling test - sodium metabisulfite): rapid screening for the presence of HbS - positive if HbS present (trait or disease) - does not distinguish trait from homozygous form → always confirm by HPLC or electrophoresis.
- Adult normal values: HbA: 96–98 %; HbA2: 2.0–3.5 %; HbF: <1.0 % (up to 2 % in some healthy adults - benign hereditary persistence of HbF); HbS, HbC, HbD, HbE: 0 % (absent in a normal adult)
- Interferences and limitations: recent transfusion ((3–4 months): Transfused erythrocytes (normal donor HbA) dilute the recipient's HbS or HbF, leading to an underestimation of abnormal fractions. Mention any recent transfusions to the lab. Infants <6 months: HbF is still predominant. Interpretation differs from adults. Normal neonatal profile: HbF + HbA ± trace HbA2. Severe iron deficiency: underestimates HbA2 (false negative for beta-thalassemia minor). Correct iron deficiency before concluding HbA2 is normal. HbA1c (glycated hemoglobin): fraction visible on HPLC before HbA. Do not confuse with an abnormal fraction.
Interpretation of Profiles and Clinical Implications
| Profile / Diagnostic | HPLC results and mechanism | Clinical implications and management |
|---|---|---|
| Normal adult profile HbA 96–98 % / HbA2 2–3,5 % / HbF <1 % |
HbA (α₂β₂): major adult fraction — physiological carrier of O₂ with normal affinity; HbA2 (α₂δ₂): minor fraction — uncertain physiological role — key biological marker of beta-thalassemia (increased if β synthesis deficit); HbF (α₂γ₂): minimal physiological persistence (<1 %) in adults — increases again in certain pathologies (sickle cell disease treated with hydroxyurea, leukemia, aplastic anemias) and in cases of benign hereditary persistence of HbF (HPFH); normal neonatal profile at birth: HbF 70–90 %+ HbA 10–30 % + HbA2 traces — γ→β switching is completed around 6 months (HbA becomes the majority) | A normal electrophoretic profile in an adult excludes a structural hemoglobinopathy and beta-thalassemia — however, it does not exclude alpha-thalassemia (mild alpha-thalassemias — alpha trait −α/αα or −−/αα — do not alter the adult electrophoretic profile → diagnosis by molecular biology — PCR specific for alpha deletions); if persistent microcytic hypochromic anemia with a normal electrophoretic profile and normal ferritin → consider alpha-thalassemia → order alpha-globin molecular genetics (LSPQ or reference laboratory); interpretation within the clinical context is always essential — a normal profile does not exclude all hemoglobinopathies. |
| Sickle cell trait (AS carrier) HbA 55–60 % / HbS 38–45 % / HbA2 2–3.5 % |
Point mutation β6Glu→Val (GAG→GTG) on an HBB gene allele → substitution of glutamic acid by valine at position 6 of the beta chain → HbS; heterozygote (healthy carrier): one normal HBB allele (→ HbA) + one mutated HBB allele (→ HbS) → majority HbA (55–60 %) + minority HbS (38–45 %); HbS polymerizes only in conditions of severe hypoxia — the AS carrier is asymptomatic in daily life but may experience complications at high altitude, during scuba diving, or in case of severe perioperative hypoxia; hemogram: normal or discrete microcytosis (if iron deficiency is associated) | Sickle cell trait (AS) is benign in the vast majority of cases — AS carriers lead normal lives without major restrictions. Genetic counseling: if both parents are AS carriers → 25% % risk of having an SS child (homozygous sickle cell disease — illness) with each pregnancy → prenatal diagnosis possible (chorionic villus sampling or amniocentesis + molecular biology). Rare clinical exceptions: splenic infarction at high altitude (>2,500 m) + recurrent hematuria (renal papillary necrosis) + risk of sudden death during prolonged intense physical exercise in heat and dehydration (case reports) — advice for caution during intensive competitive sports. Partner screening: systematically recommended if AS carrier and wishing to conceive. |
| Homozygous sickle cell disease (SS) HbS 85–95 % / HbF 2–20 % / HbA2 2–4 % / HbA 0 % |
Two mutated HBB alleles (β6Glu→Val) → no HbA produced → almost exclusive HbS; under deoxygenation conditions → HbS molecule polymerization → erythrocyte sickling (sickle cells) → microvascular obstruction (vaso-occlusion) + chronic hemolysis + endothelial inflammation activation; HbF (α₂γ₂) inhibits HbS polymerization — HbF level is a major prognostic factor (HbF >20 %→ fewer crises); hydroxyurea increases HbF synthesis by 15 to 25 % → reduction in vaso-occlusive crises; typical HPLC profile: HbS 85–95 % + variable HbF (2–20 % ) + HbA2 2–4 % + total absence of HbA | Severe chronic multisystem disease - specialized hematology follow-up mandatory; acute manifestations: painful vaso-occlusive crisis (PVD) + acute chest syndrome (ATS - life-threatening emergency - hypoxemia + pulmonary infiltrate + chest pain) + stroke (CVA - child) + splenic sequestration + aplastic crisis (parvovirus B19); chronic manifestations: splenomegaly + functional asplenia from childhood → prophylaxis with penicillin V + vaccinations (pneumococcal PCV20 + PPV23, meningococcal, Hib, influenza, COVID); disease-modifying treatments: hydroxyurea (Hydrea) 15-35 mg/kg/d → 1st line - HbF ↑ → crises ↓ - reimbursed RAMQ; L-glutamine (Endari); voxelotor (Oxbryta); crizanlizumab (Adakveo); hematopoietic stem cell (HSC) transplantation: only potentially curative treatment - indication in children with severe disease if HLA-identical donor available; gene therapy: advanced clinical trials (Lovo-cel - BLA approved FDA 2023) |
| Beta-thalassemia minor (beta-thal trait) HbA2 >3,5 % / HbF 1–5 % / HbA 92–95 % |
A mutated HBB allele (point mutation — >200 known mutations — defect in transcription, splicing, or translation of β-globin mRNA) → decreased synthesis of β chains → relative excess of α chains → increased HbA2 (compensation by δ-globin overexpression) → HbA2 >3.5% %(main diagnostic criterion) + slightly increased HbF (1–5% % ) + moderate microcytic hypochromic anemia (Hb 10–13 g/dL) + microcytic hypochromic erythrocytes + elevated erythrocyte count (relative polycythemia) + decreased MCV (60–70 fL); the carrier is generally asymptomatic or paucisymptomatic (slight fatigue) — qualified as «minor» thalassemic or thalassemic trait carrier | Frequent diagnostic trap: beta-thalassemia minor is often confused with iron deficiency (same biological picture — hypochromic microcytosis); distinguishing features: normal or high ferritin (no iron deficiency) + HbA2 >3.5 % on electrophoresis + high red blood cell count (relative polycythemia) + absence of response to iron supplementation; caution: concomitant iron deficiency lowers HbA2 → can distort diagnosis (false negative) → correct iron deficiency before electrophoresis if ferritin is low; genetic counseling: if both parents are carriers → 25 % risk of beta-thalassemia major (Cooley's disease) → severe transfusion-dependent hemolytic anemia from the first months of life → prenatal diagnosis recommended; iron supplementation is useless and potentially harmful in thalassemic carriers without confirmed deficiency |
| Other important profiles HbC, HbE, HbD, SC double heterozygotes, and Sβ-thal |
HbC (β6Glu→Lys) : fréquente en Afrique de l'Ouest — trait AC asymptomatique — maladie CC rare (anémie hémolytique modérée + splénomégalie + cristaux d'HbC dans les hématies) ; HbSC : double hétérozygote S/C — maladie drépanocytaire de sévérité intermédiaire — HbS 50 % + HbC 50 % — rétinopathie proliférative + ostéonécrose fréquentes — syndrome thoracique aigu possible ; HbSβ+-thalassémie : double hétérozygote S/β+-thal — HbS 60–85 % + HbA 10–30 % + HbA2 élevée + HbF variable — phénotype drépanocytaire modéré à sévère ; HbSβ0-thalassémie : double hétérozygote S/β0-thal — HbS 85–92 % + HbA 0 % + HbA2 élevée + HbF variable — phénotype aussi sévère que la drépanocytose SS ; HbE (β26Glu→Lys) : très fréquente en Asie du Sud-Est — trait AE asymptomatique — EE légère — HbEβ0-thal (double hétérozygote) : anémie sévère transfuso-dépendante dans les formes graves ; HPFH (persistance héréditaire de l'HbF) : HbF 15–35 % + HbA normale + asymptomatique — bénin | SC sickle cell disease accounts for about 25–30 % of sickle cell syndromes in Quebec (in patients originating from West Africa and the Caribbean)—a milder disease than SS but with significant specific complications: proliferative retinopathy (mandatory annual ophthalmological examination from the age of 10—risk of FFA + laser photocoagulation if neovascularization) + osteonecrosis of the femoral head (MRI if hip pain) + priapism + thromboembolism (increased risk compared to SS); HbE is the most widespread globin mutation in the world (100–150 million carriers)—frequent in patients originating from Cambodia, Thailand, Vietnam, and Myanmar—to consider in front of unexplained microcytosis in a patient from Southeast Asia; always interpret electrophoresis based on the patient's geographic origin |
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Alpha-thalassemia - electrophoresis limit: Unlike beta-thalassemia, alpha-thalassemia minor (α-trait - one or two deletions in the four alpha-globin genes) does not typically alter the adult electrophoretic profile—normal HbA2, normal HbF, no abnormal fraction. It manifests solely as hypochromic microcytosis with normal ferritin. Diagnosis relies on molecular biology (PCR with detection of alpha-globin deletions—−α3.7, −α4.2, −−SEA, −−MED, etc.). This limitation is clinically important: a normal electrophoresis does not rule out alpha-thalassemia in a microcytic patient without iron deficiency, particularly in patients of Asian, African, or Mediterranean origin.
Sickle cell emergencies — recognize and act fast
In a patient with known sickle cell disease (SS, SC, Sβ-thalassemia), consult immediately to the emergency room if : Chest pain, cough, fever, hypoxemia Acute chest syndrome (ACS) - a life-threatening emergency - emergency blood transfusions + antibiotics + O₂; ; Sudden focal neurological deficit (hemiplegia, aphasia, visual impairment) → Sickle cell ischemic stroke — urgent thrombolysis and/or transfusion; ; Abdominal pain + rapidly progressive splenomegaly + sudden pallor (child) → acute splenic sequestration — urgent transfusion; ; Fever >101.3 °F in a child with sickle cell disease → pneumococcal sepsis (functional asplenia) — immediate IV antibiotics.
Consult at Clinique Omicron
The doctors at Clinique Omicron prescribe and interpret hemoglobin electrophoresis, provide genetic counseling for at-risk couples, ensure first-line follow-up for carriers of hemoglobinopathies, and coordinate specialized hematology follow-up for symptomatic cases. The analyses are available from our service points in Quebec and via telemedicine. To make an appointment, visit cliniqueomicron.ca.
The content of this page is for informational purposes only and does not substitute for the advice of a qualified healthcare professional or a laboratory medicine specialist. Any diagnosed hemoglobinopathy requires appropriate medical follow-up and genetic counseling if parental planning is considered.
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