Haematology SC079: Many Member Of The Family Have Anaemia Flashcards
Structure of Hb molecule
- Globin chains x4
- β chain x2
- α chain x2 - Heme x4 (within each globin chain)
- Porphyrin
- Fe atom (Ferrous ion (+2 state) 99%, Ferric ion (+3 state) <1%)
Abnormal haemoglobin
Mutations of genes encoding Globin chains of Hb molecule
Effect:
1. Decreased production of globin chains (Thalassaemia)
- Predominantly a quantitative defect in globin chain synthesis of Hb molecule
- α thalassaemia
- β thalassaemia
—> Globin chain imbalance (more imbalance —> smaller MCV)
—> Excess globin chains is toxic to red cell precursors in BM (some will be proteolysed by RBC precursor, remaining form tetramer, premature RBC death (self notes))
-
Globin chains with abnormal function (Haemoglobinopathy)
- Predominantly a qualitative defect of production of structurally abnormal globin / Hb molecule
- Abnormal globin structure —> Abnormal Hb functions
—> Polymerisation / Aggregation of Hb causing abnormal shape + reduced solubility (e.g. Sickle cell anaemia)
—> Unstable structure (not floating properly —> Hb precipitate in RBC —> shorten t1/2 —> haemolytic anaemia)
—> Increased O2 affinity
—> Decreased O2 affinity
—> Methaemoglobinaemia (maintain in Fe3+ instead of Fe2+ state —> cannot bind O2) - Both (Thalassaemic haemoglobinopathy)
Thalassaemia
α Thalassaemia:
- Mutations involving α globin gene
- 4 α globin gene loci, located in Chromosome 16
- α globin gene mainly affected by deletions, but mutations (somatic point mutation) may also be found
β Thalassaemia:
- Mutations involving β globin gene
- 2 β globin gene loci, located in Chromosome 11
- β globin gene mainly affected by mutations (somatic point mutation), but deletions may also be found
- β0 mutations: No β chain produced
- β+ mutations: β chain production much reduced
Thalassaemia major:
- α Thalassaemia: all 4 α genes deleted
—> Hydrops fetalis (Hb Bart’s: 4γ globin (very high O2 affinity)): die in utero / after birth, may survive if active measures taken (e.g. intrauterine transfusion, intrauterine cord blood transplantation)
- β Thalassaemia: both β genes mutated (β0/β0 (homozygous) or β0/β+ (compound heterozygous))
—> Cooley’s anaemia: develop anaemia in 3-6 months after birth, transfusion dependent for life
- Require lifelong regular transfusion
Thalassaemia intermedia:
- α Thalassaemia: 3 α genes deleted
—> HbH disease (4β globin —> precipitate out —> HbH inclusion bodies)
- β Thalassaemia: both β genes mutated (β+/β+)
- Moderate anaemia: NOT require transfusion by definition, Hb 6-10, Mild jaundice (∵ premature RBC death + haemolysis), Mild-Moderate splenomegaly (∵ extramedullary haematopoiesis + haemolysis)
Thalassaemia trait:
- α Thalassaemia: 1 / 2 α genes deleted
- β Thalassaemia: 1 β genes mutated (either β0 or β)
- Mild / No anaemia: Hb 10-13, Mild / No jaundice, No splenomegaly usually (only clue is ***low MCV)
Thalassaemia trait / intermedia
- DDx of hypochromic microcytic anaemia
- DDx of mild splenomegaly (for intermedia)
- Increased ferritin in some cases
- Family screening is needed when one member is diagnosed to have thalassaemia
- Pre-natal diagnosis needed for pregnancy in at-risk couples
- When anaemia is more severe than expected from thalassaemia —> MUST investigate for other causes of anaemia
vs Fe deficiency anaemia:
1. Hb
- Trait: 10-13 (Normal / Slight ↓)
- Fe deficiency: Any level (Can be very low)
- RBC count
- Trait: Normal / ↑
- Fe deficiency: ↓ - MCV
- Trait: ↓ but usually not <65
- Fe deficiency: Any level (Can be very low) - Serum Fe
- Trait: Normal
- Fe deficiency: ↓ - TIBC
- Trait: Normal
- Fe deficiency: ↑ - % Fe saturation
- Trait: Normal
- Fe deficiency: ↓ - MCH
- Trait: ↓
- Fe deficiency: ↓ - RDW
- Trait: Normal / Slightly ↑
- Fe deficiency: Can be very high
Severe thalassaemia:
- Exactly same as Fe deficiency (∵ BM cannot compensate —> dyserythropoiesis)
- Very low Hb, Low RBC, Low MCV, Low MCH, Very high RDW
Haemoglobinopathies
- Polymerisation / Aggregation of Hb causing abnormal shape + reduced solubility
Sickle cell anaemia:
- HbS
—> β globin chain in 6th amino acid change from Glutamate to Valine
—> polymerise in hypoxic condition + reduced solubility
—> sickling of RBC (very rigid —> block capillary —> infarction / stroke)
- Occurs in people with black ancestry
- Not found in Chinese - Unstable structure (uncommon)
Haemoglobin Koln:
- Unstable Hb chains that unfold + precipitate
—> Heinz body (aggregates of precipitated unstable haemoglobin) + Irregularly contracted RBC
—> Haemolytic anaemia (may be triggered + exacerbated by infective episodes) - Increased O2 affinity
- Less likely to released bound O2
- Causes physiological erythrocytosis - Decreased O2 affinity
- SiO2 ↓, with patient relatively asymptomatic (∵ can still release O2 to tissue) - Methaemoglobinaemia
- Mutations lead to Fe in Heme ring being maintained in Fe3+ state
- MetHb: Cannot bind O2, Blue in colour (causing Cyanosis), ↑ in presence of oxidising agents (e.g. Dapsone)
Why perform Hb study?
- Dx of anaemia of patients to ensure proper treatment
- Detection of carrier state in order to provide genetic counselling
- Genotyping in antenatal diagnosis
Common trigger for Thalassaemia testing:
- Low MCV +/- Clinical features (Pallor, Splenomegaly, Failure to thrive)
Epidemiology of Thalassaemia and Haemoglobinopathy in HK Chinese
Low MCV:
- α thalassaemia: 5%
- β thalassaemia: 3%
- HbE: 0.3%
Normal MCV:
- α thalassaemia: 3% (i.e. haematologically silent)
Globin gene families
Alpha gene family (Chromosome 16):
- α1, α2
- ζ (used in embryo)
Beta gene family:
- β
- δ
- Aγ, Gγ
- ε (used in embryo)
Embryo vs Fetus vs Adult Hb
Embryo Hb:
- ζ2ε2
- α2ε2
- ζ2γ2
Fetus Hb:
- α2γ2 (HbF)
Adult Hb:
- α2β2 (HbA) (>95%)
- α2δ2 (HbA2) (1-3%)
- α2γ2 (HbF) (<1%)
Laboratory diagnosis of α thalassaemia
- Red cell indices + Clinical features
- Hb
- MCV
- RBC
- RDW - Supravital staining for HbH inclusion bodies (β4) (excess β chains)
- Thalassaemia trait will also have HbH inclusion bodies (just very few e.g. 1 in 1000 cells)
- Thalassaemia intermedia have much more (that’s why called HbH disease) - Immunochromatographic strip test for Hb Bart’s (γ4) (excess γ chains since no α chains to bind to)
- Ab against Hb Bart on the strip
Laboratory diagnosis of β thalassaemia
- No α4 tetramers (∵ too toxic to RBC precursor —> die to BM —> cannot release RBC in blood —> extremely difficult to detect excess α chain)
- Quantitation of HbA2 + HbF by HPLC (High performance liquid chromatography) / Capillary electrophoresis
- ∵ ↑ δ + γ chain production (with ↓ β chain)
—> ↑ HbA2 >3.5% (more consistent than HbF)
—> ↑ HbF (only 50%)
Summary of algorithm for diagnosis of Thalassaemia
Patient sample
—> MCV screening
—> Supravital staining / IC strip test —> α thalassaemia
—> HPLC / Capillary electrophoresis —> β thalassaemia
Laboratory diagnosis of Haemoglobinopathy
Triggers:
1. Clinical features
- Pallor, Jaundice, Splenomegaly (∵ Haemolysis)
- Plethora (∵ Erythrocytosis)
- Cyanosis (∵ MetHb, Low SaO2)
- Laboratory findings
- Haemolysis
- Erythrocytosis
- MetHb, Low SaO2
- MCV normal (∵ NO globin chain imbalance) / ↑ (∵ reticulocytosis)
Detection of Hb variants (some variants have changes in multiple properties):
1. Change in overall charge
- Altered electrophoretic mobility
- HPLC / Capillary electrophoresis
- Change in solubility (e.g. HbS)
- Peripheral blood smear
- HbS solubility test (by adding reducing agent —> create hypoxia)
- HPLC / Electrophoresis - Change in stability
- Precipitation of unstable Hb
- Peripheral blood smear (irregularly contracted cells (∵ biten by splenic macrophage), Heinz bodies)
- Heat stability test
- Isopropanol stability test - Change in O2 affinity
- O2 saturation study by Co-oximetry —> calculate P50 (partial pressure of O2 when 50% Hb is oxygenated)
- Gel electrophoresis
Relatively common Hb variants in HK
-
HbE
- β26 Glu —> Lys
- Structurally abnormal + Reduced production due to creation of new alternative splice site —> Thalassaemic haemoglobinopathy
- Normal O2 affinity
- Mildly unstable - Hb Constant Spring
- α2 142 Stop —> Gln
- Elongated mRNA (unstable) —> Markedly reduced production globin chain
Summary of algorithm for diagnosis of Haemoglobinopathy
Clinical features
—> Peripheral blood smear (sickle cell, Heinz body etc.)
—> HPLC / Capillary electrophoresis / Electrophoresis (charge separation)
—> Hb solubility test / O2 saturation study / Hb stability test
