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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 (α22) 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