Download haemoglobin abnormalities

HAEMOGLOBIN ABNORMALITIES
Dr. Nusrum Iqbal
Normal Haemoglobin
Normal adult Hb (Hb A) has two polypeptide globin chains, the α and β chains, which
have 141 and 146 amino acids, respectively
Fetal haemoglobin (Hb F), which has two α and two γ chains,
Increasing synthesis of β chains from 13 weeks of gestation and at term there is 80%
Hb F and 20% Hb A
Switch from Hb F to Hb A occurs after birth when the genes for γ chain production are
further suppressed
Hb A2 (α22) remains at a level of about 2% throughout adult life
Abnormal Haemoglobin
Abnormalities occur in
Globin chain production (e.g. thalasaemia)
Structure of the globin chain (e.g. sickle cell disease)
Combined defects of globin chain production and structure, e.g. sickle cell βthalasaemia
Genetic defects in haemoglobin are the most common of all disorders
The Thalasaemias
The thalasaemias (Greek thalasa=sea) are anaemias originally found in people living on
the shores of the Mediteranean now affect people throughout the world
Normally there is balanced (1:1) production of α and β chains
Defective synthesis of globin genes in thalasaemia laeds to ‘imbalanced’ globin chain
production, leading to precipitation of globin chains within the red cell precursosrs and
resulting in ineffective erythropoeisis
Precipitation of globin chains in mature red cells laeds to haemolysis
β-Thalasaemia
Homozygoous β-thalasaemia, either no normal β chains are produced (β0), or β-chain
production is very reduced (β+)
There is an excess α chains which precipitate erythroblasts red cells causing ineffective
erythropoiesis and haemolysis. Excess α chains combine with whatever, β,  and  chains
are produced resulting in increased quantities of Hb A2 and Hb F and, at best small
amounts of Hb A. Heterozygous β-thalasaemia there is usually symptomless microcytosis
with or without mild anaemia
Molecular Genetics
100 gneetic defects leading to β-thalasaemia genes have been characterized. Unlike αthalasaemia, the defects are mainly point mutations rather then gene deletions
Mutations result in defects in transcription, RNA splicing and modification transdation
via frame shifts and nonsense codons producsing highly unstable β-globin which cannot
be utilized
Clinical Syndromes
Clinically β-thalasaemia can be divided into the following:
Thalasaemia
minor (or trait), the symptomless heterozygous carrier state
Thalasaemia intermedia, with moderate anaemia, rarely requiring transfusions
Thalasaemia major, with severe anaemia requiring regular transfusions
Thalasaemia Minor (Trait)
This common carrier state is asymptomatic
Anaemia is mild or absent
The red cells are hypochronic and microcytic with a low MCV and MCH it may be
confused with iron deficiency in thalasaemia triat the serum freitin and the iron stores are
normal
Hb electroporesis usually shows a raised Hb A2 and often a raised Hb F
Iron should not be given to these patients unless they develop coincidental iron
deficiences
Thalasaemia Intermedia
Thalasaemia intermedia includes patients who are symptomatic with moderate anaemia
(Hb 7-10 g dl-1) and who do not require regular treansfusion
It is more severe than in β-thalasaemia trait but milder than in transfusion=dependent
thalasemia major
May be due to a combination of homozygous mild β+-and α=thalasaemia reduced α
chain precipitation and less ineffectrive erythropoiesis and haemolysis
Patients may have splenomegaly and bone deformities. Recurrent leg ulcers, gallstones
and infections are also seen
Thalasaemia major (Cooley’s anaemia)
Children affected by severe β-thalasaemia present during the first year of life with:
Failure to thrive and recurrent bacterial infections
Severe anaemia from 3-6 months when the switch from - to β-chain production should
normally occur
Extramedullary haemopoiesis that soon leads to hepatosplenomegaly and bone
expansion giving rise to the classical thalasaemic facies
Skull x-rays in these children show the characteristic ‘hair on end’ appearance of bony
trabeculation
The expansion of the bone marrow is also shown in an x-ray of the hand
Investigations
Blood count: shows a moderate to severe anaemia with reduced MCV and MCH.
Retriculocyte count is raised and nucleated red cells are present in the peripheral blood.
WCC and the number of platelets are normal unless hypersplenism is present
Blood film: shows a hypochronic and predominanatly microcytic picture.
Posplenectomy features will be present after splenectomy has been carried out
Investigations
Saturated iron-binding capacity and high serum ferritin levels: are caused by multiple
blood transfusions
Hb electrophoresis: shows an increase in Hb F, markedly reduced or absent Hb A, Hb
A2 is normal or slightly increases
Management
The aims of treatment are to suppress ineffective erythropoiesis, prevent bony
deformities and allow normal activity and development. Long term folic acid
sypplements are required regular transfusions should be given to keep the Hb above 10 g
dL-1
Blood transfusions may be required every 4-6 weeks. Febrile transfusion reaction can
be prevented by the use of leucocyte-depleted blood
Management
If transfusion requirements increase, splenectomy should be considered although this is
usually delayed until after the age of 6 years because of the risk of infection.
Prophylaxis against infection is required for pateitns undergoing splenectomy
Management
Iron overload caused by repeated transfusions (transfusion haemosidresis) may lead to
damage to the endocrine glands, liver, pancreas and the myocardium by the time patients
reach adolescence. The iron-chelating agent of choice remains deferrioxamine to be
administered parenterally
Management
Desferrioxamine is given as an overnight subcutaneous infusion on 5-7 nights each
week. Ascorbic acid 200 mg daily is given, as it increases the urinary excretion of iron in
response to desferrioxamine
With current therapy, normal growth and sexual development occur but compliance
may be a problem, especially in teenagers
Management
Excessive doses of desferrioxamine may cause cataracts, retinal damage, nerve
deafness. Infection with Yerisinilia enterocollitica occurs in iron-loaded patients treated
with deferrioxamine
Bone marrow transplantation has been used in young patients with HLA-matched
siblings
 β-Thalasaemias, Hb lepoare and hereditary persistence of fetal haemoglobin (HPFH)
These variants are due to deletions of the α- and β-globin genes and produce a milder
form of thalasaemia than homozygous β0-thalasaemia because the reduced β-chain
production is partially compensated by increased -chain synthesis
α-Thalasaemia
Molecular genetics
α-thalasaemia is caused by gene deletions. Gene for α chains is duplicated on both
chromosomes 16;i.e. there are four genes. Deletion of one α-chain gene (α+) or both αchain genes (α0) on each chromosomes 16 may occur
If all four genes are absent (deletion of both genes on both chromosomes) there is no αchain synthesis and only Hb Barts (4) is present Hb Barts cannot carry oxygen and is
incompatible with life
α-Thalasaemia
Infants are either stillborn at 28-40 weeks or die very shortly after birth. They are pale,
edematous and have enornous livers and spleens – a condition called hydrops fetalis
If three genes are deleted, there is moderate anaemia (Hb 7-10 g dL-1) and splenomegaly
(Hb H disease). The patients are not usually transfusion-dependent.
Hb A, Hb Barts and Hb H (β4) are present. Hb A2 is normal or reduced
α-Thalasaemia

If two genes are deleted (α-thalasaemia trait) there is microcytosis with or without mild
anaemia. Hb H bodies may be seen on staining a blood film with brilliant cryesyl blue.
With one gene deletion the blood picture is usually normal
Globin chain synthesis studies for the detection of a reduced ratio of α to β chains may
be necessary for the definitive diagnosis of α-thalasaemia trait
α-thalasaemia may result from genetic defects other then deletions, mutations in the stop
codon producing an α chain with many extra amino acids (Hb Constant Spring)
Sickle Syndrome
The most important structural abnormality of the Hb chain is sickle cell haemoglobin
(Hb S). Hb S results from a single-base mutation of ademine to thymine which produces
a substitution of valine for glutamine at the sixth codon of the β-globin chain. In the
homozygous state (sickle cell anaemia) both genes are abnormal (Hb SS)
Sickle Syndrome
Whereas the heterozygous state (sickle cell trait, Hb AS) only one chromosome carries
the gene. As the synthesis of Hb F is normal, the disease usually does not manifest itself
until the Hb F decreases to adult levels at about 6 months of age
The disease occurs mainly in Africans (25% carry the gene) but also found in India, the
middle East, Southern Europe
Pathogenesis
Deoxygeneted Hb S molecules are insoluble and polymerize. The fexibility of the cells
is decreased and they become rigid and take up their characteristic sickle appearance.
This process in initially reversible but, with repeated sickling, the cells eventually lose
their membrane fexibility remain in the sickle form
Pathogenesis
Sickling can produce:
A shortened red cell survival
Impaired passage of cells through the microcirculation leading to obstruction of small
vessels and tissue infarction
Sickling is precipitated by infection, dehydration, cold, acidosis or hypoxia
Adhesion proteins on activated endothelial cells may play role, particularly in vasoocclusion
Hb S relseases its oxygen to the tissues more easilty than does normal Hb
Sickle Cell Anaemia
Symptoms vary from a mild asymptomatic disorder to a severe haemolytic anaemia
recurrent severe painful crises
Condition may present in childhood with anaemia and mild jaundice. Hand-and-foot
syndrome due to infarcts of small bones is quite common in children and may result in
digits of varying lengths
In the older patient, vaso-occlusive problems occur owing to sickling in the small
vessle sof any organ, mimicking many medical and surgical emergencies

Sickle Cell Anaemia
Typical infarctive sickle crises include:
Bone pain (most common)
Chest-p;euritic pain
Cerebral-hemiparesis, fits
Kidney – papillary necrosis causing haematuria, renal tubular defect resuting in lakc of
concentration of the urine
Spleen – painful infarcts
Penis-priapsim
Liver-pain with abnormal biochemsitry
Long-term Problems
Susceptibility to infections, particularly to Streptococcus pneumoniae, which can cause
a fatal meningitis or pneumonia. Osteomylitis can occur in necrotic bone often due to
salmoneela
Ghronic leg ulcers, due to ischaemia
Gallstones; pigment stones from persistent haemolysis
Aseeptic necrosis of bone, particularly of the femoral heads
Blindness, due to retinal detachment and/ or proliferative retinopathy
Chronic renal disease
Sickle Cell Anaemia
Attacks of pain with low-grade fever last from a few hours to a few days. During a
crisis Hb does not fall unless there is one or more of the following:
Aplasia – due to decreased erythropoiesis, associated with viral infections, particularly
parvoviurs
Acute sequestration – the liver and spleen become engorged with sickle cells
Haemolysis – due to drugs, acute infection or assoicated G6PD deficiency
Investigations
Blood count: the level of Hb is in the range 6-8 g dL-1 with a high reticulocyte count
(10-20%)
Blood films can show features of hyposplenism
Sickling of red cells on a blood film can be induced in the presence of sodium
metabisulphite
Sickle solubility test: a mixture of Hb S in a reducing solution such as sodium
dithionite gives a turbid appearance due to precipitation of Hb S, where as normal Hb
gives a clear solution
Investigations
Hb Electrophoresis is always neede dto confirm the diagnosis. There is no Hb A, 8095% Hb SS, and 2-20% Hb F
The parents: of the affected child will show features of sickle cell trait
Management
The ‘steady state’ anaemia requires no treatmetn.
Precipitating factors should be avoided or treated quickly. Acute attacks require
supportive therapy with intravenous fluids, oxygen, antibiotics and adequate analgesia.
Prophylaxis is given to prevent pneumococcal infection. Folic acid is givne to pregnant
women and those with sever haemolysis
Management
Regular transfusions are given only if there is svere anaemia or if patients are having
frequent crises in order to suppress the production of Hb S. Before elective operations
and during pregnancy, repeated transfusions may be used to reduce the proportion of
circulating Hb SD to less than 20% to prevent sickling. Exchange transfusions may be
necessary in patients with severe or recurrent crises before emergency surgery.
Transfusion and splenectomy may be life-saving for young children with splenic
sequestration
Management
Hydroxyurea increases Hb F production by an unknown mechanism and reduces the
frequency of painful crises, but there is a variable response and blood counts need to be
checked every two weeks to detect myelotoxicity
Results of haemopoietic cell transplantation for patients with HLA-identical siblings
and severe disease are improving gene therapy may be possible in the future
Prognosis
Some patients with Hb SS die in the first few years of life form either infection or
episodes of sequestration.
There is marked individual variation in the severity of the disease some patients have a
relatively normal life-span with few complications
Sickle Cell Trait
The individuals have no symptoms unless extreme circumstances cause anoxia, such as
flying in non-pressurized aircraft or problems with anaesthesia. Anaesthesia should
always be carried out with care to avoid hypoxia. Sickle cell trait protects against
Plasmodium falcipanum malaria
Typically there is 60% Hb A and 40% Hb S. the blood count and film are normal. The
diagnosis is made by a positive sicke test or by Hb electrophoresis
Other Structural Globin Chain Defects
There are many Hb variants (e.g. Hb C, D) many of which are not associated with
clinical manifestiaonts.
Hb C disease may be associated with Hb S (Hb SC disease)
Clinical course is similar to that with Hb SS, but there is an increased likelihood of
thrombosis this may laed to life-threatening episodes of thrombosis in pregnacy and
retinopathy
Combined Defects of globin chain production and structure
Abnormalities of Hb structure (e.g. Hb, S, C) can occur in combination with
thalasaemia. Combination of β-thalasaemia trait and sickle cell trait (sickle cell βthalasaemia) resembles sickle cell anaemia (Hb SS) clinically.
Hb E is the most common Hb variants in South East Asia,
Homozygous Hb E causes a mild microcytic anaemia, but the combination of Hb E and
β-thalasaemia produces the clinical and haematological features of β-thalasaemia major
Prenatal diagnosis of severe haemoglobin abnormalities
Of the offspring of parents who both have either β-thalasaemia or sickle cell trait, 25%
will have β-thalasaemia major or sickle cell anaemia, respectively
Recognition of these heterozygous states in parents and family counselling provides a
basis for antenatal diagnosis
Prenatal diagnosis of severe haemoglobin abnormalities
If a pregnant woman is found to have a haemoglobin defect, her partner should be
tested. Antenatal diagnosis is offered if both are affected as there is a risk of a severe fetal
Hb defect, particularly β-thalasaemia major.
Fetal DNA analysis can be carried out using amniotic fluid, chorionic villus or fetal
blood samples.
Abortion is offered if the fetus is found to be affectec
Chorionic villus biopsy has the advantage that it can be carried out in the first trimester,
thus avoiding the need for second trimester abortions
Gene Therapy
The ultimate corrective therapy for severe Hb abnormalities would be gene therapy
Inserting normal Hb genes into the patient’s haemopoietic cells in vitro and then
transplanting these cells abck into the patient after ablative treatment had been given to
remove the abnormal bone marrow
Metabolic disorders of the red cell
Red cell metabolism
The mature red cell has no nucleus, mitochondria or ribosome therefore unable to
synthesize proteins.
Red cells have only limited enzymes systems but they are of major importance in
maintaining the viabliity and function of the cells.
Energy is required in the fomr of ATP for the maintenance of the flexibility of the
membrane and the biconcave shape of the cells to allow passage through small vassels for
regulation of the sodium and potassium pumps to ensure osmotic equilibrium
Metabolic disorders of the red cell
Red cell metablosim
It is essential that Hb be maintained in the reduced state
Enzyme systems
The glycolytic (Embden-Meyerhof) pathway
Hexose monophosphate (pentosephosphate) pathway, which provides reducing power
for the red cell in the form of NADPH
Metabolic disorders of the red cell
Red cell metabolism
90% of glucose is metabolized by the former 10% by the latter
Glutathione is important in combating oxidative stress to the red cell, and failure of this
mechanism may result in:
Rigidity due to cross-linking of spectrin, which decreases membrane felxibility causes
‘leakiness’ of the red cell membrane
Oxidation of the Hb molecule, producing methaemoglobin precipitation of globin
chains as Heinz bodies localized on the inside of the membrane
Glucose-6-phosphate dehydrogenase (G6PD) deficiency
The enzyme G6PD holds a vital position in the hexose monophosphate shunt oxidizing
glucose-6-phosphate to 6-phosphogluconate with the reduction of NADP to NADPH
G6PD deficiency is a common condition that presents with a haemolytic anaemia and
affects millions of people throughout the world, particularly in Africa, around the
Mediteranean, the Middle East and South East Asia
The gene for G6PD is sex-linked
The deficiency therefore affects males
Glucose-6-phosphate dehydrogenase (G6PD) deficiency
Heterozygotes have some protection against Plasmodium falcipanum
There are over 400 structural types of G6PD, and mutations are mostly single amino
acid substitutions
There are many variatns with reduced activity but only two are common. In the
African, or A- type, the degree of deficiency is mild more makred in older cells
Glucose-6-phosphate dehydrogenase (G6PD) deficiency
Haemolysis is self limiting as the young red cells newly produced by the bone marrow
have nearly normal enzyme activity.
However in the Mdeiterranean type, both young and old red cells have very low
enzyme activity.
After an oxidant shock the Hb level may fall precipitously; death may follow unless the
condition is recognized and the patient is transfused urgently
Clinical Syndromes
Acute drug-induced haemolysis
Favism (ingestion of fava beans)
Chronic haemolytic anaemia
Neonatal jaundice
Infections and acute illness will also percipitate haemolysis in patients with G6PD
deficiency
the clinical features are due to rapid intravascular haemolysis with symptoms of
anaemia, jaundice and haemoglobinuria
Investigations
Blood Count: is normal between attacks
During an attack: the blood film may show irregularly contracted cells, bite cells (cells
with an indentation of the membrane, blister cells (cells in which the Hb appears to have
become partially detached from the cell membrane; heinz bodies (best seen on films
stained with methyl voilet) and reticulocytosis
Investigations
Haemolysis: is evident
G6PD deficiency: can be detected using several screening tests, such as demonstration
of the decreased ability of G6PD-deficient cells to reduce dyes.
The level of the enzyme may also be directly assayed.
Treatment
Any offending drugs should be stopped
Underlying infection should be treated
Blood transfusion may be life-saving
Splenectomy is not usually helpful
Pyruvate kinase deficiency
This is the most common defect of red cell metabolism after G6PD deficiency,
affecting thousands rather than millions of people.
There is reduced production of ATP causing rigid red cells
Homozygotes have haemolytic anaemia and spenomegaly
It is inherited as an autosomal recessive
Investigations
ANaemia: of variable severity is present (Hb 5-10 g dL-1). The oxygen dissociation
curve is shifted to the right as a result of the rise in intracellular 2,3-DPG and this reduces
the severity of symptoms due to anaemia
Blood film: shows distorted (‘prickle’) cells and a reticulocytosis
Pyruvate kinase activity: is low (affected homozygotes have levels of 5-20%)
Treatment
Blood transfusion may be necessary during infections and pregnancy
Splenectomy may improve the clinical condition and is usually advised for patients
requiring frequent transfusions