Haemoglobin is a tetramer of two pairs of globin chains. Each chain encloses one haem group containing iron in the ferrous (Fe²⁺) state. [1–4]

Each tetramer therefore binds up to four O₂ molecules, one at each haem iron. Oxygen binds the haem irons—not the central cavity of the tetramer.

Fe²⁺ binds oxygen reversibly. Oxidation to Fe³⁺ creates methaemoglobin, which cannot bind oxygen normally.

The fetal γ→adult β switch explains two classic findings:

• β-thalassaemia major usually presents after 3–6 months, when HbF falls and β-chain demand rises.
• In α-thalassaemia, excess γ chains form Hb Bart’s (γ₄) before/around birth, whereas excess β chains form HbH (β₄) later.

The β-like cluster is ordered broadly in the sequence of developmental expression: embryonic ε → fetal γ → adult δ/β. Regulatory-region lesions can therefore reduce globin expression even when a structural gene remains intact.

• In α-thalassaemia, excess β chains can form β₄ tetramers (HbH). HbH is unstable and has abnormally high O₂ affinity, but some reaches the circulation.
• In β-thalassaemia, excess α chains cannot form a stable tetramer. They precipitate early in erythroid precursors, causing profound ineffective erythropoiesis.

The traditional labels major, intermedia, and trait remain useful for teaching, but genotype does not map perfectly to clinical severity. Current practice also uses:

• Transfusion-dependent thalassaemia (TDT)
• Non-transfusion-dependent thalassaemia (NTDT)

Regular transfusion decisions depend on repeated haemoglobin values plus growth, symptoms, organ effects, and ineffective erythropoiesis—not genotype alone. ^[12]

α-thalassaemia is usually caused by deletion of one or more of the four α-globin genes on chromosome 16.

• Cis: --/αα; both missing genes are on one chromosome. Common in Southeast Asian/Chinese populations.
• Trans: -α/-α; one missing gene on each chromosome. More common in some African and Mediterranean populations.

The blood count may look similar, but reproductive risk differs. Two cis α⁰ carriers can conceive a fetus with --/-- and Hb Bart’s hydrops.

With only one working α gene, excess adult β chains form HbH (β₄):

• Very high O₂ affinity → impaired tissue O₂ unloading
• Unstable → precipitation and inclusion formation
• Chronic haemolysis → anaemia, jaundice, splenomegaly, pigment gallstones
• Oxidative stress or infection may worsen haemolysis

With no α genes, the fetus cannot make normal HbF because HbF also requires α chains. Ineffective erythropoiesis and haemolysis produce profound fetal anaemia, while excess γ chains form Hb Bart’s (γ₄), which holds oxygen too tightly to unload it effectively. The combination of severe anaemia and poor tissue oxygen delivery causes high-output cardiac failure and hydrops. Survival is possible only with intensive fetal and postnatal treatment and remains highly specialised.

Hb Constant Spring results from a stop-codon mutation in the α2 gene, producing an elongated, unstable α chain in very low quantity. HbH–Constant Spring can be more severe than deletional HbH disease.

β-thalassaemia usually results from point mutations affecting transcription, splicing, translation, or mRNA processing:

• β⁰: no β-chain output from the affected allele
• β⁺: reduced, but not absent, β-chain output

1. Reduced β-chain synthesis leaves excess unpaired α chains.
2. α chains precipitate in erythroid precursors → ineffective erythropoiesis.
3. Surviving abnormal red cells undergo haemolysis.
4. Anaemia drives EPO and massive marrow expansion.
5. Extramedullary haematopoiesis produces hepatosplenomegaly.
6. Ineffective erythropoiesis suppresses hepcidin → increased intestinal iron absorption.
7. Regular transfusion adds further iron with no physiological excretion pathway.

• Pallor, fatigue, jaundice, pigment gallstones
• Failure to thrive and delayed puberty
• Frontal bossing, maxillary expansion, “hair-on-end” skull from marrow expansion
• Hepatosplenomegaly and hypersplenism
• Iron loading of the heart, liver, pituitary, pancreas, and other endocrine organs
• Cardiac iron overload → arrhythmia and cardiomyopathy

• Which relatives are affected, and what is the inheritance pattern?
• Ethnic and geographic ancestry as a probability modifier, never an exclusion rule
• Age of onset and childhood growth
• Transfusion history and chelation adherence
• Jaundice, dark urine, gallstones, episodic crises
• Pregnancy plans and previous affected pregnancies
• Drugs or oxidant exposure
• Symptoms of cardiac, hepatic, or endocrine iron overload

• Pallor and scleral icterus
• Splenomegaly/hepatomegaly
• Growth and pubertal development
• Frontal bossing or maxillary prominence
• Signs of heart failure, chronic liver disease, diabetes, or hypogonadism
• Bronze/grey skin pigmentation in iron overload

• HPLC separates haemoglobins by interaction with a charged column; retention time and peak area identify and quantify fractions.
• Capillary electrophoresis (CE) separates haemoglobins by electrophoretic mobility.
• A retention window or migration zone suggests identity but is not always definitive because variants may co-elute or co-migrate.

• Low MCV/MCH with normal or high RBC count
• HbA₂ >3.5%
• HbF may be mildly raised

Iron deficiency can sometimes suppress HbA₂ and complicate a borderline result. Interpret the pattern with iron status and the clinical context.

Adult α-thalassaemia trait often has:

• Microcytosis with normal iron status
• Normal HbA₂ and HbF
• No reliably detectable abnormal fraction on routine HPLC/CE

Useful tests include:

1. Supravital HbH inclusion-body stain: brilliant cresyl blue precipitates unstable HbH into multiple fine “golf-ball” inclusions. Numerous positive cells support HbH disease; rare cells may occur in trait. A negative result does not exclude trait.
2. Neonatal Hb Bart’s immunochromatographic test: detects γ₄; the result supports α-chain deficiency but does not alone define genotype.
3. α-globin molecular analysis: definitive when carrier genotype and reproductive risk matter.

• Gap-PCR: common deletions such as --SEA
• ARMS-PCR/reverse dot blot: known point mutations
• Sequencing: uncommon or novel variants

Indications include an atypical phenotype, uncertain variant identity, discordant routine results, and prenatal diagnosis.

HbS results from β6 Glu→Val. Deoxygenated HbS polymerises, producing rigid sickled cells:

• Vaso-occlusion → pain crises, acute chest syndrome, stroke, priapism
• Haemolysis → jaundice, gallstones, anaemia
• Repeated splenic infarction → functional asplenia

Triggers include hypoxia, dehydration, acidosis, infection, cold, and altitude. HbS is uncommon in ethnic Chinese populations, but ancestry must not be used to exclude it.

The HbS solubility test becomes turbid when deoxygenated HbS precipitates. It does not distinguish trait from disease and is unreliable when HbS concentration is low, including newborns or recently transfused patients; confirm with Hb fractionation.

Unstable globin unfolds and precipitates as Heinz bodies, damaging the membrane and causing extravascular haemolysis. Infection or oxidative stress can precipitate a crisis.

• Supravital stain: one or a few coarse Heinz bodies
• Film: bite cells or irregularly contracted cells
• Heat/isopropanol test: abnormal precipitation

Do not confuse:

• Heinz bodies: denatured haemoglobin
• HbH inclusions: many fine deposits producing a golf-ball appearance
• Howell–Jolly bodies: nuclear remnants in hyposplenism/asplenia

• HbE (β26 Glu→Lys) creates an alternative splice site, causing both an abnormal β chain and reduced output—a thalassaemic haemoglobinopathy. HbE/β-thalassaemia can be severe.
• Hb Constant Spring is an elongated, unstable α chain produced in very low quantity. In combination with α⁰-thalassaemia, it causes HbH–Constant Spring disease.

A left-shifted dissociation curve prevents tissue unloading:

1. Tissues sense relative hypoxia.
2. EPO rises.
3. Compensatory erythrocytosis develops.

P50 is reduced. Consider this when erythrocytosis is familial and JAK2-negative.

A right-shifted curve promotes O₂ release. The patient may have cyanosis or low measured saturation yet remain relatively well because tissue delivery is preserved. P50 is increased.

Fe³⁺ haem cannot carry oxygen normally.

• Congenital: HbM variants or cytochrome b5 reductase deficiency
• Acquired: oxidants such as dapsone, nitrites, benzocaine, primaquine, and aniline dyes
• Clues: cyanosis poorly responsive to O₂, chocolate-brown blood, saturation gap, pulse oximetry tending toward ~85%
• Confirm with co-oximetry
• Acquired symptomatic disease may be treated with methylene blue, but this may be ineffective or hazardous in G6PD deficiency

• Sickle cell disease is autosomal recessive; disease-modifying care may include hydroxyurea, vaccination and infection prevention, plus prompt management of vaso-occlusive and acute chest crises.
• Some unstable haemoglobins and high- or low-affinity variants are autosomal dominant, so a vertical family history may be present.
• For unstable haemoglobins, avoid recognised oxidant triggers and treat precipitating infection; severe or recurrent haemolysis requires specialist management.
• Management of altered-affinity variants is phenotype-specific. Establish the variant before treating an otherwise unexplained familial erythrocytosis or cyanosis.

• Reassure: it is usually clinically benign.
• Avoid unnecessary iron treatment.
• Treat proven coexisting iron deficiency.
• Document the diagnosis to prevent repeated investigation.
• Offer partner/family screening and genetic counselling where relevant.

• Specialist monitoring of growth, bone health, thrombosis risk, pulmonary pressure, splenomegaly, and extramedullary haematopoiesis
• Intermittent or regular transfusion only for defined clinical indications
• Monitor iron loading even without transfusion because low hepcidin increases absorption
• Chelate when indicated by cumulative assessment
• Consider splenectomy only in selected cases after weighing infection, thrombosis, and pulmonary-hypertension risks

• Regular red-cell transfusions; a usual pre-transfusion target is approximately 9–10.5 g/dL, individualised
• Iron monitoring using serial ferritin plus liver/cardiac MRI when indicated
• Iron chelation
• Allogeneic haematopoietic stem-cell transplantation for eligible patients
• Gene-addition or gene-editing therapies for selected patients where available
• Multidisciplinary cardiac, hepatic, endocrine, fertility, bone, and infection care

HbA1c may be unreliable in thalassaemia because haemolysis shortens red-cell survival, transfused donor cells alter the measured population, and some variants interfere with particular assays. Compare HbA1c with capillary or plasma glucose data; when discordant, use an appropriate alternative such as fructosamine or continuous glucose monitoring.

Investigate rather than attributing everything to thalassaemia:

• Iron, folate, or B12 deficiency
• Bleeding
• Infection or inflammation
• Parvovirus B19 aplastic crisis
• Renal disease
• Hypersplenism
• Another haemolytic process

Haemoglobin studies are performed to:

1. Diagnose anaemia correctly.
2. Detect carriers for counselling.
3. Define familial variants for prenatal or preimplantation testing.

When one person is diagnosed, screen the partner and relevant first-degree relatives with CBC, iron status, and Hb analysis; add molecular testing when α-thalassaemia or an uncertain variant remains relevant.

For two carriers of the same autosomal-recessive severe condition, each pregnancy classically has:

• 25% affected
• 50% carrier
• 25% unaffected/non-carrier

This calculation applies only after the parental genotypes are defined. Not every pairing of “thalassaemia carriers” creates the same fetal risk.

With an experienced operator, the additional miscarriage risk from CVS or amniocentesis is likely below 0.5%; use current local counselling. ^[10]

A transfusion-dependent thalassaemia patient with EF 15% and poor medication adherence most likely has iron-overload cardiomyopathy from inadequate chelation.