Unit 1: Haemoglobin chemistry
Basic Haematological Techniques-II notes · PTU syllabus (BMLS402-18)
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Unit summary
Haemoglobin carries oxygen, and its abnormal forms cause disease. This unit covers haemoglobin pigments and their measurement, and the identification and estimation of abnormal haemoglobins.
After this unit you can
- Describe the structure and types of normal haemoglobin
- Measure haemoglobin derivatives such as methaemoglobin and carboxyhaemoglobin
- Identify abnormal haemoglobins
- Estimate HbF and HbA2
PTU syllabus topics
Haemoglobin pigments and their measurement, abnormal haemoglobins — identification and estimation
Oxyhaemoglobin
Hb carrying oxygen
Bright red
Methaemoglobin
Iron in the ferric (Fe³⁺) state
Can't carry oxygen; some drugs and nitrites
Carboxyhaemoglobin
Hb bound to carbon monoxide
CO poisoning: cherry-red colour
Sulphaemoglobin
Sulphur bound to Hb
Irreversible; some drugs
Topic 1
Structure and normal haemoglobins
- Haemoglobin: a tetramer of four globin chains, each with a haem group (protoporphyrin + ferrous iron Fe²⁺) binding one O₂ molecule; molecular weight about 64,500.
HbA
α₂β₂
About 97%
HbA2
α₂δ₂
1.5–3.5%
HbF (fetal)
α₂γ₂
Less than 1% (70–90% at birth); higher oxygen affinity
- Embryonic haemoglobins: Gower 1, Gower 2 and Portland in early pregnancy.
Topic 2
Haemoglobin pigments (derivatives)
Oxyhaemoglobin
Hb + O₂
Normal arterial form; bright red
Deoxyhaemoglobin
O₂ released
Venous blood
Methaemoglobin
Iron oxidised to Fe³⁺; cannot carry O₂
Drugs (dapsone, nitrites), hereditary; cyanosis with chocolate-brown blood; normal under 1–2%
Carboxyhaemoglobin
Hb + CO (affinity 200–250 times O₂)
Carbon monoxide poisoning; cherry-red colour; smokers up to 10%
Sulphaemoglobin
Sulphur bound irreversibly
Drugs; persists for the red cell lifespan
Cyanmethaemoglobin
Methaemoglobin + cyanide
Stable form used to measure total Hb
Topic 3
Measuring haemoglobin derivatives
- Spectrophotometry: each pigment has characteristic absorption peaks — oxyhaemoglobin at 541 and 577 nm, methaemoglobin at 630 nm (disappears on adding cyanide — Evelyn–Malloy method), carboxyhaemoglobin at 538 and 568 nm. CO-oximeters in blood gas analysers measure all fractions automatically.
- Simple tests: alkali denaturation colour test for carboxyhaemoglobin (stays pink with NaOH), chocolate colour of methaemoglobin that does not turn red on shaking with air.
Topic 4
Abnormal haemoglobins (haemoglobinopathies)
Sickle cell disease (HbS)
β6 glutamic acid → valine; polymerises when deoxygenated
Sickled cells, haemolytic anaemia, painful crises; common in central India tribal populations
HbC, HbD, HbE
Other β-chain substitutions
Mild anaemia; HbE common in north-east India
β-Thalassaemia
Reduced or absent β-chain synthesis
Major: severe transfusion-dependent anaemia; trait: microcytosis with raised HbA2
α-Thalassaemia
Reduced α-chains
HbH (β₄) and Hb Bart's (γ₄)
Topic 5
Identification and estimation
- Sickling test
- Blood with 2% sodium metabisulphite under a sealed coverslip; sickled cells appear in 30 minutes to 24 hours
- Solubility test
- HbS insoluble in dithionite phosphate buffer — turbid
- Haemoglobin electrophoresis
- Cellulose acetate pH 8.6: order from anode — HbA, HbF, HbS, HbA2/C
- HPLC (cation exchange)
- Quantifies HbA2, HbF and variants — method of choice
- HbF estimation
- Alkali denaturation (Betke) — HbF resists alkali; Kleihauer–Betke stain shows HbF cells
- HbA2 estimation
- Column chromatography or HPLC; above 3.5% suggests β-thalassaemia trait
- NESTROFT
- Naked-eye single-tube red cell osmotic fragility test — screening for thalassaemia trait
Example
Mentzer index (MCV ÷ RBC count) below 13 suggests thalassaemia trait; above 13 suggests iron deficiency.
Key terms
- Haem
- Iron-porphyrin group binding oxygen
- Methaemoglobin
- Haemoglobin with ferric iron that cannot carry oxygen
- Carboxyhaemoglobin
- Haemoglobin bound to carbon monoxide
- Haemoglobinopathy
- Inherited disorder of haemoglobin structure or synthesis
- HbA2
- Minor adult haemoglobin raised in β-thalassaemia trait
Quick revision
- HbA, HbA2, HbF; embryonic haemoglobins.
- Oxy-, deoxy-, met-, carboxy-, sulph- and cyanmethaemoglobin.
- Spectral peaks; Evelyn–Malloy; CO-oximetry.
- HbS, HbC, HbD, HbE; α and β thalassaemia.
- Sickling, solubility, electrophoresis, HPLC, HbF and HbA2 estimation, NESTROFT, Mentzer index.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Give the chain composition of HbA, HbA2 and HbF.
- Q2.Why is carbon monoxide poisonous?
- Q3.What causes methaemoglobinaemia?
- Q4.What is the mutation in HbS?
- Q5.How is the sickling test performed?
- Q6.What does a raised HbA2 indicate?
Long-answer questions
- Q1.Describe normal haemoglobins and haemoglobin derivatives.
- Q2.Describe methods of measuring methaemoglobin and carboxyhaemoglobin.
- Q3.Describe the laboratory diagnosis of sickle cell disease and thalassaemia.
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