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
