Haemoglobin structure and function

Structure

Tetramer: two pairs of globin chains, each with a haem (protoporphyrin IX + Fe2+)
One Hb binds 4 O2; ~640 million molecules per red cell

zeta/epsilon -> alpha/gamma (HbF) by ~10 weeks -> alpha/beta (HbA) from ~32 weeks
HbF ~70-90% at birth, falls to <2% by 12 months
- Explains why beta-chain disorders (sickle, beta-thalassaemia) present at 3-6 months, not at birth, while alpha-thalassaemia presents in utero
Switch controlled by BCL11A (the target of gene therapy - exagamglogene autotemcel)

Haemoglobinopathies are the commonest monogenic disorders worldwide - ~5% of the global population carry a significant variant
Carrier frequencies track historical malaria distribution
- Alpha-thalassaemia: South-East Asia, Southern China, Pacific, Africa
- Beta-thalassaemia: Mediterranean, Middle East, Indian subcontinent, South-East Asia
- HbS: sub-Saharan Africa, Middle East, India
- HbE: the commonest variant in South-East Asia - relevant to Australia's migrant population
>400 structural variants described; most clinically silent

Alpha cluster - chromosome 16 (zeta, alpha2, alpha1)
- Four alpha genes (two per chromosome) -> dose-dependent phenotype
Beta cluster - chromosome 11 (epsilon, Ggamma, Agamma, delta, beta)
- Two beta genes -> heterozygote vs homozygote

Cooperativity - O2 binding shifts T (tense, low affinity) -> R (relaxed, high affinity)
- -> sigmoid dissociation curve
Right shift = reduced affinity = more O2 delivered to tissue
- CADET: inc CO2, inc Acid (dec pH), inc 2,3-DPG, inc Exercise, inc Temperature
Left shift = higher affinity, less release
- HbF (does not bind 2,3-DPG -> pulls O2 across the placenta), carboxyhaemoglobin, methaemoglobin, hypothermia, alkalosis, stored blood (2,3-DPG depleted)
Bohr effect - CO2/H+ lower affinity in tissue; Haldane effect - deoxy-Hb carries more CO2

Mechanism	Example
Reduced chain synthesis	Thalassaemias -> imbalanced chains -> unstable tetramers -> ineffective erythropoiesis
Structural variant	HbS (beta Glu6Val) - polymerises when deoxygenated -> sickling
Unstable Hb	Heinz bodies, congenital Heinz body anaemia
Altered affinity	High-affinity -> erythrocytosis; low-affinity -> cyanosis with normal PaO2
Oxidised iron (Fe3+)	Methaemoglobinaemia - cannot bind O2, left-shifts the rest

First-line

FBE + film - MCV, MCH, RDW, target cells, basophilic stippling, sickle cells, Heinz bodies
Ferritin - exclude iron deficiency before interpreting HbA2
HPLC or capillary electrophoresis - quantifies HbA, HbA2, HbF and variants
DNA analysis for alpha-thalassaemia (deletional - not detected by electrophoresis)

Pattern	Diagnosis
HbA2 >3.5%	Beta-thalassaemia trait
Low MCV/MCH, normal HbA2 and HbF, normal ferritin	Alpha-thalassaemia trait (diagnosis of exclusion -> DNA)
HbS on electrophoresis, sickle solubility positive	Sickle cell trait or disease
HbF markedly raised	Beta-thalassaemia major/intermedia, HPFH, juvenile CML
HbH inclusions on brilliant cresyl blue	HbH disease (3 alpha genes deleted)

A microcytosis with a normal or high red cell count and a normal RDW suggests thalassaemia trait, not iron deficiency (Mentzer index MCV/RBC <13)
Beta-thalassaemia trait can be masked by coexisting iron deficiency - HbA2 falls. Repeat after iron repletion.

Genes deleted	Phenotype
1 (-a/aa)	Silent carrier
2 (-a/-a or --/aa)	Trait - microcytosis, no anaemia
3 (--/-a)	HbH disease - moderate haemolytic anaemia
4 (--/--)	Hb Barts hydrops fetalis - lethal in utero

Co-oximetry for met- and carboxyhaemoglobin (pulse oximetry is unreliable; SpO2 sticks near 85% in methaemoglobinaemia)
p50 for high/low-affinity variants
HbA1c is invalid in the presence of a haemoglobinopathy -> use OGTT or fructosamine

Antenatal screening in at-risk populations; partner testing if the woman is a carrier
Pre-implantation and prenatal diagnosis where both partners carry
Carriers need to know their status - it is a reproductive issue, not a health problem

Transfusion-dependent: regular transfusion to pre-transfusion Hb ~90-100 g/L + iron chelation (deferasirox, deferoxamine, deferiprone)
- Monitor iron by *T2 cardiac and R2 liver MRI**, not ferritin alone
Non-transfusion-dependent: avoid unnecessary transfusion, monitor for iron loading from increased gut absorption
Luspatercept - reduces transfusion burden in transfusion-dependent beta-thalassaemia
Allogeneic HSCT is curative; gene therapy/BCL11A editing available in selected centres
Folate; avoid routine iron supplementation

Hydroxyurea/hydroxycarbamide - raises HbF, reduces crises and mortality
Transfusion/exchange for stroke, acute chest syndrome, pre-operatively
Penicillin prophylaxis and full encapsulated-organism vaccination (functional asplenia)
Voxelotor, crizanlizumab, L-glutamine; curative gene therapy and HSCT

Methylene blue 1-2 mg/kg IV if symptomatic or metHb >20-30%
- *Contraindicated in G6PD deficiency* -> ascorbic acid, exchange transfusion

Avoid oxidant drugs (dapsone, primaquine, nitrates, local anaesthetics) in unstable Hb and G6PD deficiency
Genetic counselling referral for every new carrier identified

Malaria resistance - the selective pressure behind all common variants
HbE/beta-thalassaemia - the commonest severe thalassaemia syndrome in South-East Asia
Sickle cell trait: renal medullary carcinoma, papillary necrosis, exertional rhabdomyolysis, splenic infarction at altitude
High-affinity Hb variants -> familial erythrocytosis with a low EPO
Low-affinity variants -> cyanosis with a normal PaO2 and normal cardiac studies
Acquired HbF elevation: juvenile myelomonocytic leukaemia, aplastic anaemia, pregnancy, hydroxyurea
Alpha-thalassaemia with myelodysplasia (ATMDS, somatic ATRX)
HPFH - benign; important not to mistake for thalassaemia

Carriers (trait) are healthy - normal life expectancy, mild microcytosis only
- The harm done is iatrogenic: years of unnecessary iron therapy and endoscopy
Hb Barts hydrops fetalis - fatal in utero or neonatally; maternal risk of pre-eclampsia and obstructed labour
HbH disease - variable; haemolysis, gallstones, splenomegaly, crises with oxidant stress or infection
Beta-thalassaemia major - presents at 3-6 months with failure to thrive; without transfusion, death in childhood
- With transfusion + chelation, survival into the 5th-6th decade; iron-overload cardiomyopathy is the leading cause of death
- Extramedullary haematopoiesis, frontal bossing, "hair-on-end" skull, pathological fracture
- Endocrinopathy from iron: hypogonadism, diabetes, hypothyroidism, hypoparathyroidism, growth failure
Sickle cell disease: median survival now into the 5th-6th decade in high-income settings; chronic organ damage dominates

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