W2L3 - Haemoglobinopathies Flashcards
Adult Haemoglobin Components
HbA - 97% HbA2 - 2%-3% HbF - <1% Composed of - HbA = α2β2 - HbA2 = α2δ2 - HbF = α2γ2
How are the individual haemoglobin ‘types’ assessed?
Electrophoresis
HPLC
Isoelectric focussing
Analytical Effects
In some of these disorders the RBC may be resistant to lysis by reagents employed by automated haematology analysers (AHA)
In this circumstance, routine FBC results may be affected…
- typically RBC are lysed to allow analysis of WBC
- => failure to lyse RBC => falsely increased [WBC]
- often ‘flagged’ by the analyser
Repeat analysis with extended lysis (‘lysis resistant mode’) usually rectifies the situation
Laboratory Assessment of Hb - Electrophoresis
At alkaline pH, haemoglobin is a negatively charged protein and will migrate toward the anode (+)
Structural variants that have a change in the charge on the surface of the molecule at alkaline pH will separate from HbA
Haemoglobin variants that have:
- an amino acid substitution that is internally sited may not separate
- an amino acid substitution that has no effect on overall charge will not separate by electrophoresis
Laboratory Assessment of Hb - HPLC
HPLC depends on the interchange of charged groups on the ion exchange material with charged groups on the haemoglobin molecule
When a haemolysate containing a mixture of haemoglobins is adsorbed onto the resin, the rate of elution of different haemoglobins is determined by the pH and ionic strength of any buffer applied to the column
Elution of the charged molecules is achieved by a continually changing salt gradient
Fractions are detected as they pass through an ultraviolet/visible light detector and are recorded
Analysis of the area under these absorption peaks gives the percentage of the fraction detected
The time of elution (retention time) of any normal or variant haemoglobin present is compared with that of known haemoglobins, providing quantification of both normal haemoglobins (A, F and A2) and many variants
Disorders of Haemoglobin
Disorders of Haemoglobin may result from:
- altered Hb structure
- altered Hb stability
- altered Hb production
- altered Hb amount
- altered Hb function
Disorders of Hb - Thalassemias and Haemoglobinopathies
Thalassemias - group of recessively autosomal inherited diseases characterised by a decreased or absent synthesis of one of the two globin chains that form adult haemoglobin (haemoglobin A, α2β2)
Haemoglobinopathies - group of inherited disorders that result in the altered structure of haemoglobin
Clinical Features of Haemoglobinopathies
Very variable Anaemia variable mild-severe - lethargy, weakness etc. - jaundice Medullary haematopoiesis - bone deformation Extramedullary haematopoiesis - hepato-splenomegaly, ‘other tissue masses’
Thalassemia
Mutations or deletions of the alpha or beta globin genes lead to reduced synthesis of alpha or beta globin chains
Alpha thalassemia results from mutations in alpha globin gene
Beta thalassemia results from mutations in the beta globin gene
The resultant anaemia in thalassemia is due to the deleterious effects of excessive (unbalanced) production of the unaffected globin chains
Alpha Thalassemia
A tandem pair of alpha globin genes (chromosome 16) is inherited from each parent => yielding four alpha globin genes
If one or more alpha globin genes are non-functional => alpha thalassemia
Reduced alpha globin synthesis leads to an imbalance in alpha & non alpha (beta, gamma, delta) globin synthesis
- reduced haemoglobin synthesis
- microcytic, hypochromic RBC
- shortened half-life of RBC due to formation of unstable homotetramers of excess non-alpha globin chains may lead to RBC destruction in the bone marrow
Alpha Thalassemia - Clinical Manifestations
Dependent on how many (of 4) genes affected
- 1 gene: (- α /αα) alpha thalassemia minima
- 2 genes: (- α/-α “trans” or –/ αα “cis”) alpha thalassemia minor/ alpha thalassemia trait
- 3 genes (–/-α) HbH disease
- 4 genes: (–/– ) Hydrops fetalis
Alpha Thalassemia Minimia (- α/αα)
Typically clinically unapparent Normocytic, normochromic No HbH inclusion Normal HbA2 & HbF as adults ‘Silent carrier’
Alpha Thalassemia Minor (-α/-α or –/ αα)
Mild anaemia Microcytic, hypochromic RBC Target cells/poikilocytes Occasional HbH inclusion Normal HbA2 & HbF as adults α/β ratio ~ 0.8/1.0
Alpha Thalassemia - HbH Disease (–/-α)
Results in moderately severe haemolytic anaemia as HbH is unstable
- microcytic, hypochromic
- poikilocytosis, target cells
- precipitated haemoglobin evident with supravital stains
Alpha Thalassemia - Hydrops Fetalis (–/– )
Prevents formation of all normal haemoglobins (haemoglobins F, A, and A2)
In this situation, the major haemoglobin that forms in the foetus is haemoglobin Bart’s, a non-functional gamma chain homotetramer (γ4)
Oxygen delivery to foetal tissues is markedly reduced, leading to a severe hypoxic condition
Beta Thalassemia
A single β gene is located on each chromosome 11
Most β thalassemias result from point mutations in the β globin gene
Nomenclature:
- β+ => partial block in β-chain synthesis
- β0 => complete absence of β-chain synthesis
Reduced beta chain synthesis leads to decreased HbA (α2β2)
Promotes compensatory increase of gamma & delta globins that leads to increased levels of HbA2 (α2δ2) & HbF (α2γ2)
Excess alpha chains form insoluble alpha chain tetramers (α4) which are destroyed in bone marrow/spleen
Beta Thalassemia Minor Features
Clinical - typically minimal clinical signs Laboratory results - mild anemia - microcytic, hypochromic Peripheral blood smear - variable anisocytosis and poikilocytosis - target cells, basophilic stippling - slightly ↑ polychromatophilic RBC Hb electrophoresis - mild increase in HbA2 (4-7% instead of normal 2-3%)
Beta Thalassemia Intermedia Features
Clinical - active erythropoiesis - splenomegaly Laboratory results - moderate anaemia - microcytic, hypochromic - ↑ reticulocytes Peripheral blood smear - target cells, basophilic stippling, nRBCs BM - erythroid hyperplasia Hb electrophoresis: 1. Severe forms - HbA2 5-10% - HbF 30-75% - HbA remainder 2. Milder forms - HbA2 >3.2% - HbF 1.5-12.0%
Beta Thalassemia Major
Beta thalassemia major (homozygous β0/β0)
Marked by severe microcytic hypochromic anaemia & anisopoikilocytosis
No beta chain is produced at either allele (β0 signifying a non-functional gene) and nearly all the hemoglobin is HbF with a small fraction of HbA2
Requiring multiple red cell transfusions
Inadequate transfusion therapy leads to:
- childhood growth retardation
- skeletal abnormalities
- hepatosplenomegaly
- high risk of infection
Beta Thalassemia Major Features
Laboratory results - severe anaemia - microcytic hypochromic - ↑ RDW Peripheral blood smear - anisocytosis, poikilocytosis - basophilic stippling, polychromasia - nRBCs Hb electrophoresis 1. Cord blood - < 2% HbA 2. Adult - β0/β0 => ‘No’ HbA, 90% HbF, variable HbA2 - β0/β+ and β+/β+ => ‘some’ HbA, majority HbF, normal to ↑ HbA2
Haemoglobinopathies
Specific inherited point mutations of the beta globin gene lead to abnormal unstable haemoglobin
- e.g. HbS, HbC, & HbE
Autosomal recessive
- symptomatic homozygous state ‘disease’
- asymptomatic heterozygous state ‘trait’
Often result in haemolytic anaemia
Sickle Cell (HbS) Disease
Substitution of 6th amino acid on β chain
Glutamic acid (polar) replaced with non polar valine
- heterozygous ααββs (sickle cell trait)
- homozygous ααβsβs (sickle cell disease)
Deoxygenation => HbS polymerisation, sickling & microvascular occlusion
Re-oxygenation of erythrocytes breaks down the HbS polymer and restores the normal shape
This process of sickling and un-sickling continues until the erythrocyte membrane is no longer flexible
Irreversibly sickled cells undergo intravascular haemolysis or extravascular removal by the reticuloendothelial system
Sickle Cell Disease Features
Laboratory findings - Hb ~ 60-90 g/L - sickle cells, poikilocytosis Solubility test (sodium dithionite) - HbS is insoluble in deoxygenated state - crystals refract light, solution becomes turbid Electrophoresis/HPLC - used to identify/quantify HbSS
HbC Disease
Predominantly west African populations Substitution of lysine for glutamine in β-globin Rhomboid haemoglobin crystals form in RBC Hb identification - HbC migrates with HbA2 Homozygous HbC (ααβc βc) - mild haemolytic anaemia, target cells Heterozygous HbC (ααββc) - benign, few target cells Blood film - microcytosis, target cells
HbE Disease
Largely confined to south east Asian populations
Homozygous HbE (ααβeβe)
- mild haemolytic anaemia
- mild microcytic, hypochromic
Combination of HbE β-thalassemia (HbE/β0 ) => severe disease, usually as severe as β-thalassemia major
Molecular Diagnostics for Haemoglobinopathies
Multiplex gap-PCR
- primer spacing too great for normal allele
- product only for deletion allele
Direct sequencing
- detect point mutations
Multiplex ligation dependent probe amplification (MPLA)
- quantitative amplification of multiple fluorescent probe pairs to α− & βglobin
- signal increases with duplication
- signal decreases with deletion
Microarrays
