This pathology lecture focuses on the laboratory diagnostic approach to haemoglobin disorders — specifically thalassaemias and haemoglobinopathies. It is the pathology counterpart to the clinical haematology lecture on inherited anaemias. The lecture teaches you which tests to order, why they work, and how to interpret them for carrier detection, definitive diagnosis, prenatal diagnosis, and genetic counselling in Hong Kong Chinese patients.

1. Understand the laboratory tests and their principles for diagnosing thalassaemia and haemoglobinopathy ^[1] 2. Summarise the laboratory approach to haemoglobin disorders ^[1]

• Thalassaemia is the most common genetic disease in HK — every clinician must know how to screen for it
• This lecture bridges basic science (globin gene structure) with clinical pathology (HPLC, capillary electrophoresis, molecular testing)
• Exam questions frequently ask you to distinguish thalassaemia trait from iron deficiency anaemia using CBC parameters, to identify the correct screening test for antenatal thalassaemia, and to interpret Hb pattern study results
• Directly tested in past papers: Q12 of 2022 Fourth Summative MCQ asked about the most useful screening test for thalassaemia in the antenatal period ^[3]

Three key reasons to perform haemoglobin studies: ^[1]

1. Differential diagnosis of anaemia to ensure proper treatment
2. Detection of carrier state for genetic counselling
3. Genotyping in prenatal diagnosis

Why this matters from first principles: Thalassaemia trait is microcytic like iron deficiency, but giving iron to a thalassaemia patient is not only useless — it can cause iron overload. Carrier detection is crucial because if both parents are carriers, there is a 25% chance of a severely affected child (e.g., Hb Bart's hydrops fetalis or β-thalassaemia major). Prenatal diagnosis allows informed reproductive decisions.

Carrier rates in HK Chinese: ^[1]

• α-thalassaemia: ~5% (3% with low MCV, remainder with normal MCV)
• β-thalassaemia: ~3%
• Hb E: ~0.3%

Clinical significance: With these carrier rates, the chance of two α-thal carriers meeting and having a child with Hb Bart's hydrops fetalis is significant. This is why antenatal screening with MCV is standard practice in HK.

Haemoglobin is a tetramer composed of two pairs of globin chains, each with a haem group containing iron in the ferrous (Fe²⁺) state ^[1][2]

Normal adult haemoglobin composition:

^[1]

Why this matters: All three normal haemoglobins share α-chains. Therefore:

• α-thalassaemia → reduced α-chains → affects ALL haemoglobins → excess β-chains form β₄ (Hb H) or excess γ-chains form γ₄ (Hb Bart's)
• β-thalassaemia → reduced β-chains → compensatory increase in δ and γ production → raised Hb A₂ and Hb F

α-globin gene cluster: Chromosome 16 — contains ζ, α₂, α₁ genes (4 α-gene copies total: αα/αα) ^[1] β-globin gene cluster: Chromosome 11 — contains ε, Gγ, Aγ, δ, β genes ^[1]

Key point: Because there are four α-globin gene copies but only two β-globin gene copies, α-thalassaemia has a wider clinical spectrum (silent carrier → trait → Hb H disease → hydrops fetalis) than β-thalassaemia.

Thalassaemia = globin chain imbalance ^[1]

The fundamental problem is not just reduced production of one chain — it is the accumulation of the unpaired partner chain that causes damage:

• In α-thal: excess β-chains → β₄ tetramers (Hb H) → these are unstable, precipitate in RBCs → haemolysis + ineffective erythropoiesis
• In β-thal: excess α-chains → do NOT form stable tetramers → precipitate as monomers/dimers in erythroid precursors → severe ineffective erythropoiesis → this is why β-thal major is clinically MORE severe than Hb H disease

Key distinguishing features: ^[1]

Iron deficiency anaemia smear: pencil cells (elliptocytes), hypochromic cells, anisopoikilocytosis ^[1] β-thalassaemia trait smear: target cells, more uniform microcytosis ^[1]

In severe thalassaemia, the CBC parameters converge with iron deficiency — both show very low Hb, low RBC, low MCV, low MCH, and very high RDW ^[1]

Why this matters: When the thalassaemia is severe (intermedia or major), you cannot rely on CBC alone to distinguish it from iron deficiency. You need Hb pattern studies (HPLC/CE) and possibly molecular testing.

Thalassaemia major blood smear: marked anisopoikilocytosis, target cells, teardrop cells, nucleated red blood cells, basophilic stippling ^[1]

The trigger is a low MCV ± clinical features (pallor, splenomegaly, failure to thrive) ^[1]

Approach for Laboratory Diagnosis of Thalassaemia: ^[1]

1. Patient sample → MCV screening
2. If low MCV → Supravital staining / IC strip test (for α-thal) AND HPLC / Capillary Electrophoresis (for β-thal)
3. α-thalassaemia identified OR β-thalassaemia identified

Unlike β-thalassaemia (where Hb A₂ is raised), α-thalassaemia does not cause a characteristic change in Hb A₂ level on HPLC. The diagnosis relies on detecting the abnormal haemoglobin products of excess β or γ chains.

Supravital staining demonstrates Hb H (β₄) inclusion bodies ^[1]

• α-thal trait: rare Hb H inclusions (golf-ball-like inclusions in a few RBCs)
• α-thal intermedia / Hb H disease: abundant Hb H inclusions (many RBCs affected)

Principle: When blood is incubated with brilliant cresyl blue, the unstable β₄ tetramers precipitate within the red cells, forming characteristic golf-ball or mulberry-like inclusion bodies visible under light microscopy.

Why this works: In α-thalassaemia, the excess β-chains form Hb H (β₄), which is unstable and precipitates. The more α-genes deleted, the more Hb H produced, and the more inclusions seen.

Newer method: IC strip test for Hb Bart's (γ₄) ^[1]

Principle: In neonates with α-thalassaemia, excess γ-chains form γ₄ tetramers (Hb Bart's). This is detectable at birth using a rapid IC strip test on cord blood.

Why γ₄ and not β₄ in neonates: At birth, the predominant non-α chain is γ (fetal haemoglobin). So excess non-α chains in neonatal α-thal form Hb Bart's (γ₄). After the globin switch in infancy, the excess non-α chains become β, forming Hb H (β₄).

In β-thalassaemia, decreased β-chain production leads to compensatory increased δ and γ chain production → increased Hb A₂ and Hb F ^[1]

Why Hb A₂ goes up: When β-globin production falls, there are "spare" α-chains looking for partners. The δ-globin gene is on the same chromosome cluster as β, and its expression increases slightly to mop up some excess α-chains → more α₂δ₂ = more Hb A₂. Similarly, γ-chain expression increases → more α₂γ₂ = more Hb F.

Two main methods for quantitating Hb A₂ and Hb F: ^[1]

1. High Performance Liquid Chromatography (HPLC)
2. Capillary Electrophoresis (CE)

HPLC principle: Separates haemoglobin variants based on their charge differences. The sample is passed through a column with cation-exchange resin. Different Hb variants elute at different times, producing characteristic peaks. The area under each peak gives the percentage.

Capillary Electrophoresis principle: Separates haemoglobins by their electrophoretic mobility in a capillary filled with buffer. Faster, more automated, and increasingly used alongside HPLC.

Triggers include clinical features (pallor, jaundice, splenomegaly, plethora, cyanosis) AND laboratory findings (haemolysis, erythrocytosis, methaemoglobinaemia/low SaO₂) ^[1]

Note the key difference from thalassaemia: Thalassaemia workup is triggered by low MCV. Haemoglobinopathy workup is triggered by clinical features + lab abnormalities such as haemolysis, polycythaemia, or unexplained cyanosis.

Hb E (β26 Glu→Lys): ^[1]

• Reduced production due to creation of a new alternative splice site
• Normal oxygen affinity
• Mildly unstable

Hb Constant Spring (α2 termination codon 142, Stop→Gln): ^[1]

• Markedly reduced production due to unstable mRNA

Why Hb E is a "thalassaemic haemoglobinopathy": The Glu→Lys mutation at codon 26 creates an abnormal mRNA splice site that diverts much of the pre-mRNA away from normal splicing → reduced β-globin output, mimicking β-thal. When combined with a β-thal allele (Hb E/β-thal), the result can be as severe as β-thalassaemia major — this is the most common severe thalassaemia worldwide.

Why Hb Constant Spring matters: The stop codon mutation allows translation to continue into the 3' UTR, producing an elongated α-chain. The mRNA is highly unstable → very low α-chain production. Compound heterozygosity (--SEA/αCSα) produces Hb H–Constant Spring disease, which is more severe than typical Hb H disease.

Four properties that can be altered in Hb variants: ^[1]

1. Change in overall charge → detected by electrophoresis/HPLC/CE
2. Change in solubility → Hb S solubility test
3. Change in stability → heat/isopropanol stability test
4. Change in oxygen affinity → P50/oxygen saturation study

Diagnostic approach: ^[1]

1. Patient sample → Peripheral blood smear
2. HPLC / Capillary Electrophoresis
3. Further electrophoresis if needed
4. Special tests based on clinical suspicion: Hb solubility test, Hb stability test, O₂ saturation study

Indications for molecular analysis: ^[1]

1. To investigate patients with atypical phenotypes
2. To confirm identity of Hb variant
3. Use in prenatal diagnosis

α-thalassaemia: ^[1]

• (--SEA) deletion: 90% of α-thal alleles in HK Chinese

β-thalassaemia point mutations: ^[1]

• Codons 41-42 (-CTTT): β⁰ — 46%
• IVS-II-654 (C→T): β⁰ — 28%
• nt -28 (A→G): β⁺ — 13%
• Codon 17 (A→T): β⁰ — 6%

β⁰ vs β⁺ explained:

• β⁰: Complete absence of β-globin production from that allele (e.g., frameshift, nonsense, or splice site mutations that completely abolish production)
• β⁺: Reduced (but not absent) β-globin production (e.g., promoter mutations like nt -28)
• Homozygous β⁰/β⁰ → β-thalassaemia major
• β⁺/β⁺ or β⁺/β⁰ → may be thalassaemia major or intermedia depending on the residual output

Two approaches: ^[1]

1. Quick PCR-based methods for common mutations — gap-PCR for --SEA deletion, ARMS-PCR or reverse dot-blot for known point mutations
2. Direct nucleotide sequencing for uncommon/novel mutations

Methods for prenatal diagnosis: ^[1]

1. Preimplantation genetic diagnosis (PGD) — testing embryos during IVF before implantation
2. Chorionic villous biopsy (CVS) — 10–12 weeks gestation
3. Amniocentesis — 15–18 weeks gestation
4. Fetal DNA in maternal plasma — non-invasive prenatal testing (NIPT), increasingly used

Why CVS is preferred over amniocentesis: CVS can be performed earlier (10–12 weeks vs 15–18 weeks), allowing earlier decision-making. The risk of miscarriage is similar (~0.5–1%).

NIPT for thalassaemia: Cell-free fetal DNA in maternal plasma can be analysed for paternal mutations. If the fetus has NOT inherited the paternal mutation, no further invasive testing is needed. If the paternal mutation IS detected, CVS/amniocentesis may still be needed to determine if the fetus also inherited the maternal mutation.

The 2022 Fourth Summative MCQ Q12 asked: "A 20-year-old woman attended antenatal clinic at 14 weeks of gestation. She had a family history of thalassaemia and her complete blood count showed microcytic anaemia. Which is the MOST USEFUL screening test for thalassaemia in the antenatal period?" ^[3]

Answer: C. Haemoglobin pattern study (i.e., HPLC/CE for Hb A₂ quantitation)

Why not MCV? MCV is the initial screening test that flags potential thalassaemia carriers, but MCV is NOT specific — it cannot distinguish iron deficiency from thalassaemia. The question states she already HAS microcytic anaemia, so MCV has already been done. The haemoglobin pattern study is the next step that confirms whether the microcytosis is due to thalassaemia (raised Hb A₂) or not.

The 2021 MCQ Q28 presented a transfusion-dependent thalassaemia patient with heart failure and EF 15% with poor medication compliance. Answer: B. Haemochromatosis — each unit of blood contains ~250 mg iron; without chelation therapy, iron accumulates in the heart causing dilated cardiomyopathy ^[4][5].

The 2020 MCQ Q31 presented a 25-year-old woman with mild microcytic anaemia (Hb 10.7, MCV 78) and isolated severe thrombocytopenia (33 × 10⁹/L). Answer: C. Thalassaemia intermedia with hypersplenism — the microcytic anaemia suggests thalassaemia, and the isolated severe thrombocytopenia with otherwise normal WBC is best explained by hypersplenism from the enlarged spleen seen in thalassaemia intermedia ^[6].

From senior notes ^[5]:

• Iron chelation agents:

• Indicated when ferritin > 1000 μg/L or positive liver/cardiac MRI T2*
• Venesection for hereditary haemochromatosis (not for transfusion-dependent patients who are already anaemic)

From senior notes ^[7]: Thalassaemias cause minimal splenomegaly (1–2 cm) — β-thalassaemia intermedia/major and Hb H disease. In contrast, CML, myelofibrosis, and malaria cause massive splenomegaly.

From the clinical haematology companion lecture ^[2]:

• Inherited haemolytic anaemias are classified by the component of the RBC that is defective: membrane (hereditary spherocytosis), enzyme/metabolism (G6PD, pyruvate kinase), or haemoglobin (thalassaemias, sickle cell, unstable Hb)
• The pathology lecture provides the laboratory methods to characterise the haemoglobin-related causes

1. "A 25-year-old pregnant woman with MCV 72 fL. What is the most appropriate next investigation?" → Hb pattern study (HPLC/CE)

2. "How do you distinguish thalassaemia trait from iron deficiency anaemia on CBC?" → RBC count (high in thal trait, low in IDA), RDW (normal in thal trait, high in IDA)

3. "A neonate is found to have Hb Bart's on cord blood screening. What does this indicate?" → α-thalassaemia (the more Hb Bart's, the more severe)

4. "Name the most common α-thalassaemia mutation in HK Chinese." → --SEA deletion (90%)

5. "Name the two most common β-thalassaemia mutations in HK Chinese." → Codons 41-42 (-CTTT) β⁰ (46%), IVS-II-654 (C→T) β⁰ (28%)

6. "What is the diagnostic test for α-thalassaemia that is NOT detected by HPLC?" → Supravital staining for Hb H inclusion bodies / molecular testing

7. "Why is Hb A₂ raised in β-thalassaemia trait?" → Reduced β-chain production → excess α-chains pair with δ-chains → increased α₂δ₂ (Hb A₂)

• When asked for the "most useful screening test for thalassaemia": state "Haemoglobin pattern study by HPLC/capillary electrophoresis" — not just "blood test" or "MCV"
• When asked to differentiate thal trait from IDA: create a table with Hb, RBC, MCV, MCH, RDW, and iron studies
• When asked about prenatal diagnosis: mention both the invasive methods (CVS, amniocentesis) AND non-invasive (NIPT using cell-free fetal DNA)