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 β-thalasaemia

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 α-thalasaemia, 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 α-chain genes (α0) on each chromosomes 16 may occur

If all four genes are absent (deletion of both genes on both chromosomes) there is no α-chain 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 vaso-occlusion

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, 80-95% 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 β-thalasaemia) 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

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