Diagnosis and Management of Congenital Haemolytic Anaemia

CME Haematology

Diagnosis and management of Asian origin. It is characterised by the spherical shape of the affected red cells. The pattern of inheritance is autosomal congenital haemolytic anaemia dominant in 75% of cases; in the others the parents are apparently normal. HS can result from mutations in seven dif- Shameem Mahmood MBBCh BAO MRCP, levels, unconjugated hyperbilirubinaemia Specialist Registrar in Haematology and low serum haptoglobin levels. A ferent genes, but mutations in the genes coding for ankyrin, band 3 or β-pectrin David Rees FRCP FRCPath, Senior Lecturer in blood film should be reviewed; if this Paediatric Haematology shows marked fragmentation, the possi- are most common in the autosomal dominant form.3 Department of Paediatric Haematology, bility of angiopathic haemolysis, such as Clinically, HS commonly presents as King’s College London School of Medicine, disseminated intravascular haemolysis or King’s College Hospital, Denmark Hill, haemolytic uraemic syndrome, should be anaemia of variable severity, jaundice, London considered. If haemolysis is present, the which is often present neonatally, and direct antiglobulin test will identify the palpable splenomegaly (the main site of 4 Clin Med 2007;7:625–9 vast majority of cases of autoimmune red cell destruction). haemolysis; if it is negative, congenital red cell abnormalities should be considered. Diagnosis. Diagnosis of HS is based on: Congenital haemolytic anaemias are • an appropriate family history hereditary conditions resulting from an spherocytes on the blood film increase in the rate of red cell destruction.1 Cytoskeletal membrane • Four main types of red cell abnormality disorders • mean cell haemoglobin concentration which is increased in can cause haemolysis: The group of hereditary cytoskeletal approximately 50% of patients • cytoskeletal membrane disorders membrane disorders arise from muta- (eg hereditary spherocytosis (HS), tions of genes controlling the proteins of • increased osmotic fragility. hereditary elliptocytosis (HE), the membrane and their interaction Binding of the fluorescent dye EMA, as hereditary pyropoikilocytosis which results in change to the shape of measured by flow cytometry, is reduced (HPP)) the red cells.2 The inheritance pattern is in most cases of HS and can be used as a • disorders of membrane cation typically autosomal dominant. confirmatory test in atypical cases with transport (eg hereditary no family history, particularly to confirm 5 stomatocytosis) Hereditary spherocytosis the diagnosis prior to splenectomy. • enzyme deficiencies (eg dehydrogenase (G6PD) Probably the commonest cause of con- Treatment. Many patients require no spe- deficiency, pyruvate kinase (PK) genital haemolytic anaemia in northern cific treatment, although folic acid is deficiency) Europeans is HS. It is less well charac- sometimes used because of the increased terised in other populations, although it disorders of haemoglobin synthesis rate of erythropoiesis. Splenectomy sig- • certainly occurs in people of African and (eg sickle cell anaemia, unstable nificantly increases the haemoglobin and haemoglobins). Congenital haemolytic anaemia can Table 1. Useful initial investigations for possible congenital haemolytic anaemia. also be a feature of more generalised metabolic problems not directly involving Test Possible result the red cells such as lipid abnormalities Full blood count Anaemia (eg abetalipoproteinaemia, sitostero- laemia) and Wilson’s disease. Although Blood film Polychromasia, spherocytes, anisopoikilocytosis, stomatocytosis, sickle cells most thalassaemia syndromes involve an Reticulocyte count Increased element of haemolysis, the predominant defect is ineffective erythropoiesis and the Liver function tests Unconjugated hyperbilirubinaemia former will not be considered further in Serum haptoglobin Decreased this article. Lactate dehydrogenase Increased Coombs’ test (direct autoantibody test) Negative in congenital haemolysis Osmotic fragility tests Increased in presence of spherocytes General approach to diagnosis Red cell G6PD and pyruvate kinase assays Low levels in deficiency states It is first necessary to decide whether there Haemoglobin analysis Detects sickle and most other abnormal is any evidence of haemolysis, which typi- haemoglobins cally is suggested by anaemia, reticulocy- G6PD = glucose-6-phosphate dehydrogenase. tosis, high serum lactate dehydrogenase

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(a) (c) Hereditary stomatocytosis Dehydrated hereditary stomatocytosis. This is also known as hereditary xerocytosis or cryohydrocytosis. The red blood cells have a membrane abnormality, with increased permeability to cations with a greater efflux of K+ than of Na+. This causes red cell dehydration and conse- quently results in osmotically resistant xerocytes.9 The clinical presentation ranges from mild to moderate haemolytic anaemia, jaundice, splenomegaly (b) (d) and cholelithiasis. Iron overload may develop later in life. The leak of K+ from the erythrocytes can cause marked pseudohyperkalaemia, and the condition is also linked to spontaneously resolving fetal and neonatal ascites. Splenectomy is not beneficial and has been associated with increased risk of thrombosis.2

Overhydrated hereditary stomatocytosis. This disorder is also known as hydrocy- tosis. The primary defect is due to Fig 1. Blood films: (a) hereditary spherocytosis, (b) hereditary elliptocytosis, increased Na+ and K+ permeability. The (c) glucose-6-phosphate dehydrogenase deficiency, (d) sickle cell anaemia. influx of Na+ is greater than the loss of K+, resulting in a net influx of water, reduces haemolysis in most patients with Disorders of membrane cation overhydration and swelling. The HS. It is indicated if the patient requires transport resulting increased osmotic fragility and regular or frequent blood transfusions or reduced deformability of erythrocytes has significant symptoms such as failure Disorders of membrane cation transport leads to haemolysis,10 which is usually to thrive or disabling fatigue. Sympto- are characterised by red cells with a mild and does not require intervention. matic gallstones may require a cholecys- mouth-like slit rather than the normal central circular area of pallor. They typi- tectomy, and splenectomy is usually Red cell enzyme defects performed at the same time to reduce the cally involve increased leak of sodium risk of further cholelithiasis.6 and potassium across the red cell mem- Glucose-6-phosphate brane, resulting in an altered state of red dehydrogenase deficiency cell hydration and haemolysis. Diagnosis Hereditary elliptocytosis and is made on the basis of clinical suspicion The commonest inherited enzyme defi- hereditary pyropoikilocytosis and measurement of red cation transport ciency is G6DP, affecting 400 million or ektacytometry.2,8 people worldwide. The inheritance is HE is typically an autosomal dominant condition characterised by the elliptical shape of the red blood cells. It is usually Table 2. Agents likely to cause haemolytic anaemia in glucose- asymptomatic, causing minimal haemol- 6-phosphodiesterase deficiency. ysis. Occasionally it can cause severe Agent haemolysis, which is often more apparent in the newborn period. Homo- Antibiotics Sulphonamides, dapsone, co-trimoxazole, septrin, zygosity for mutations causing elliptocy- quinolones, nalidixic acid tosis typically results in HPP, a severe Analgesics Phenacetin haemolytic anaemia with bizarrely Antimalarials Maloprim, primaquine, pentaquine 7 shaped red cells. Treatment includes Other drugs Rasburicase, flutamide, methylene blue folic acid supplementation. In homozy- Acidosis Infections, fever, diabetic ketoacidosis gous HE or HPP, severe haemolytic Food Fava beans anaemia may develop and splenectomy Other Naphthalene (mothballs) may be beneficial.

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X-linked, typically resulting in affected The clinical severity varies from These are all rare and many involve only males. However, it is not uncommon to asymptomatic anaemia to a transfusion- a handful of cases worldwide. Sometimes see females with acute haemolysis due to dependent condition. Jaundice and gall- red cell enzymopathies can present as G6PD deficiency because of either stones may be present, and there is often part of a more generalised metabolic homozygosity or skewed inactivation of progressive iron overload from increased problem, often associated with neuro- the X chromosome. The condition is iron absorption and blood transfusion. A muscular problems. All can be diagnosed most prevalent in West Africa, the blood film reveals poikilocytosis and dis- by specific enzyme assay, although Mediterranean, the Middle East and torted ‘prickle’ cells. The diagnosis is studies suggest that once G6PD and PK South-East Asia, and there is some evi- made by direct enzyme assay, although deficiency have been excluded, about dence that it gives protection against significant deficiency may be missed by 70% of cases of congenital non-sphero- malaria and its complications.2 Typically, standard assays which are not sensitive to cytic haemolytic anaemia remain undi- patients are well, with no evidence of subtle variations in enzyme kinetics. agnosed despite extensive enzymopathy haemolysis unless exposed to certain Assays at low substrate concentration testing. drugs or fava beans which cause acute and different temperatures may be intravascular haemolysis. This may helpful, with DNA analysis of the PK-LR Disorders of haemoglobin present as jaundice, fever, backache gene.2,14 synthesis and pallor with dark urine due to Management depends on the severity. 11 haemoglobinuria. Intermittent or regular blood transfusions Sickle cell disease G6PD deficiency is also an important may be necessary. Splenectomy leads to a cause of prolonged or severe neonatal small increase in haemoglobin in most Disorders of haemoglobin synthesis are a jaundice, particularly when co-inherited cases. Patients should be monitored group of conditions characterised by the with Gilbert’s syndrome.2 Less com- regularly for iron accumulation. presence of sickle haemoglobin, intermit- monly, severe deficiency can result in tent vaso-occlusion and haemolysis. The chronic haemolysis. Rarer red cell enzymopathies commonest and most severe form is Diagnosis rests upon the typical his- sickle cell anaemia (HbSS), but HbS can tory, with blood tests showing intravas- There are more than 15 other red cell be co-inherited with a number of other cular haemolysis. A blood film should enzymes whose deficiency has been iden- β-globin mutations to form sickle cell show contracted and fragmented cells, tified as causing haemolytic anaemia. disease; the most common of these is with hemighosts in which the haemoglobin occupies one half of the cell. The diagnosis is confirmed by measurement Table 3. Rare red blood cell (RBC) enzymopathies. Reproduced with permission of 2 of G6PD activity in red cells. Blackwell Publishing. Management depends on the symp- RBC enzymopathy Inheritance Characteristic features toms. It is important to stop the offending drug and treat any infection. A Pyrimidine 5’ nucleotidase deficiency Autosomal recessive Basophilic stippling Mild haemolysis. blood transfusion may be needed. Renal Possible association with function should be supported as learning difficulties intravascular haemolysis can result in Hexokinase deficiency Autosomal recessive Typically mild haemolysis renal impairment. Neonatal jaundice Triose phosphate isomerase deficiency Autosomal recessive Variable haemolysis with may require phototherapy and exchange progressive neurological transfusion.12 deterioration and early death Phosphofructokinase deficiency Autosomal recessive Mild haemolysis Pyruvate kinase deficiency Pain on exercise and PK deficiency is the commonest enzy- fatigue Myoglobinuria mopathy, causing chronic, non-sphero- Typically fatal cytic haemolytic anaemia. The exact Aldolase deficiency Autosomal recessive Haemolysis prevalence is not known, but there are Myopathy probably 200–300 patients in the UK with Mental retardation the condition. The inheritance pattern is Phosphoglycerate kinase deficiency X-linked recessive Haemolysis autosomal recessive. PK catalyses the final Muscle pain step in the glycolytic pathway leading to Weakness Aphasia the accumulation of 2,3-diphosphoglyc- Myoglobinuria erate, which causes low oxygen affinity Glutathione synthetase deficiency Autosomal recessive Neurological defects and results in the anaemia being typically 5-oxoprolinuria well tolerated.13

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Key Points like peripheral blood picture with hypochromic red cells, increased formation of methaemoglobin or severe Congenital haemolytic anaemia is typically suggested by anaemia, reticulocytosis, 2 high serum lactate dehydrogenase levels, unconjugated hyperbilirubinaemia, low neonatal jaundice. haptoglobin levels and a negative direct antiglobulin test Diagnosis. Diagnosis of unstable haemo- Hereditary spherocytosis is the commonest cause of congenital haemolytic anaemia globins may be suggested by tests showing in northern Europeans decreased haemoglobin stability, such as Glucose-6-phosphate dehydrogenase deficiency is X-linked. It is more prevalent in the isopropanol or heat stability tests. West Africa, the Mediterranean, the Middle East and South-East Asia Staining with brilliant cresyl blue may show typical Heinz bodies in the red cells. The two main pathological processes in sickle cell disease are vaso-occlusion and haemolysis, both related to polymerisation of the deoxygenated haemoglobin Haemoglobin electrophoresis may detect molecule containing the mutated β-globin chain the mutant haemoglobin, but if it is very unstable it may not be detectable in the Unstable haemoglobins are typically due to point mutations causing amino acid blood. DNA analysis of the globin genes substitution in the α- or β-globin chains, the commonest probably being will show the mutation causing the Haemoglobin Köln abnormal haemoglobin. Unstable haemo- KEY WORDS: congenital haemolytic anaemia, glucose-6-dehydrogenase deficiency, globins are rare and occur sporadically; hereditary spherocytosis, sickle cell disease, unstable haemoglobins the commonest is probably Haemoglobin Köln (β98,ValMet). Haemolysis is variable and can result in transfusion HbSC. The haemoglobin S mutation is pulmonary damage, and retinopathy. It is dependence. the result of valine substituting for glu- the commonest cause of stroke in child- tamic acid at position 6 in the β-globin hood, affecting about 6% of children by Management. The response to splenec- chain. Sickle cell disease is a strikingly the age of 10 years. tomy is typically good, although only lim- variable condition; many of the con- ited experience exists for any particular tributing factors are unknown, although Management. This includes primary pre- variant. Infection and oxidative drugs higher fetal haemoglobin levels have been vention of infection with pneumococcal may precipitate acute haemolysis. linked to less severe clinical problems.15 vaccines and penicillin, with specific Gallstones may require a cholecystectomy. Both the two main pathological treatment of the complications encoun- Folic acid is usually given.17 processes in sickle cell disease, vaso- tered. There is evidence that children at occlusion and haemolysis, are related to high risk of stroke can be identified using the polymerisation of the deoxygenated transcranial Doppler scanning and that References haemoglobin molecule containing the regular blood transfusions can reduce 1 Hoffbrand AV, Pettit JE, Moss PA. Essential β mutated -globin chain. The ischaemic the risk of stroke by 90%. Hydroxyurea haematology, 5th edn. Oxford: Blackwell damage caused by vaso-occlusion has reduces the frequency of acute pain and Science, 2003:57–90. been appreciated for many years, but it acute chest syndrome. Allogeneic bone 2 Hoffbrand AV, Catovsky D, Tuddenham has recently emerged that haemolysis marrow transplantation is potentially EG. Postgraduate haematology, 5th edn. Oxford: Blackwell Science, 2005:85–150. may also contribute to some of the com- indicated in children requiring regular 3 Edelman EJ, Maksimova Y, Duru F, Altay 2,16 plications such as pulmonary hyperten- blood transfusions. C, Gallagher PG. A complex splicing defect sion, leg ulcers and priapism. Haemolysis associated with homozygous ankyrin- deficient hereditary spherocytosis. Blood causes release of free haemoglobin; this Unstable haemoglobins binds rapidly to nitric oxide (NO), 2007;109:5491–3. 4 Delhommeau F, Cynober T, Schischmanoff resulting in functional NO deficiency. Unstable haemoglobins are typically due PO et al. Natural history of hereditary This process is best studied in sickle cell to point mutations causing amino acid spherocytosis during the first year of life. disease, but is likely to contribute to the substitutions in the α- or β-globin chains. Blood 2000;95:393–7. increased risk of pulmonary hyperten- These substitutions weaken non-covalent 5 Delaunay J. The molecular basis of sion found in other conditions causing bonds, allowing the haemoglobin to hereditary red cell membrane disorders. Review. Blood Rev 2007;21:1–20. chronic haemolysis, such as pyruvate denature and precipitate as insoluble glo- 6 Bolton-Maggs PH, Stevens RF, Dodd NJ kinase and hereditary spherocytosis. bins which may attach to the cell mem- et al; General Haematology Task force of Sickle cell disease has a wide range of brane forming Heinz bodies.17 Patients the British Committee for Standards in clinical manifestations, including acute may present in various ways: congenital Haematology. Guidelines for the diagnosis and chronic pain, increased risk of cer- non-spherocytic haemolytic anaemia, and management of hereditary spherocytosis. Review. Br J Haematol tain infections due to hyposplenism, splenomegaly, pigmented gallstones, 2004;126:455–74. avascular bone necrosis, nocturia or Heinz body haemolytic anaemia with sen- 7 Gallagher PG, Forget BG. Hematologically enuresis, cholelithiasis, acute and chronic sitivity to oxidant drugs, a thalassemia- important mutations: spectrin variants in

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hereditary elliptocytosis and hereditary Von Willebrand on chromosome 12. The primary gene pyropoikilocytosis. Blood Cells Mol Dis product is a protein with a molecular 1996;22:254–8. weight of approximately 250 kDa. 8 Delaunay J. The hereditary stomatocytoses: disease genetic disorders of the red cell membrane Within cells of synthesis the vWF protein permeability to monovalent cations. Semin undergoes polymerisation, forming a Hematol 2004;41:165–72. range of multimers up to 80 protein Jonathan T Wilde MD FRCP FRCPath, 9 Carella M, Stewart G, Ajetunmobi JF et al. Consultant in Haemostasis, University units in size. vWF multimers are secreted Genomewide search for dehydrated from blood vessel endothelial cells hereditary stomatocytosis (hereditary Hospital, Birmingham NHS Foundation Trust xerocytosis): mapping of locus to directly into the circulation or are chromosome 16 (16q23-qter). Am J Hum Clin Med 2007;7:629–32 retained in the cells in specialised storage Genet 1998;63:810–6. granules called Weibel-Palade bodies 10 Fricke B, Argent AC, Chetty MC et al. The from which they are released as a ‘stomatin’ gene and protein in response to vascular injury. overhydrated hereditary stomatocytosis. Von Willebrand disease (vWD) is the Blood 2003;102:2268–77. most common of the inherited bleeding The main function of vWF is to facili- 11 Beutler E. G6PD deficiency. Review. Blood disorders, with a prevalence of sympto- tate platelet binding to blood vessel 1994;84:3613–36. matic vWD around 125 per million.1 It is subendothelium at sites of vascular 12 Dalal BI, Kollmannsberger C. Drug- caused by mutations affecting the von damage in high shear flow environments induced haemolysis and such as small arteries and the microvas- methaemoglobinaemia in glucose-6 Willebrand factor (vWF) gene which 3 phosphate dehydrogenase deficiency. Br J result in either quantitative or qualitative culature (Fig 1). Once platelets are Haematol 2005;129:291. abnormalities of vWF. anchored in place, vWF enables aggrega- 13 Beutler E, Baronciani L. Mutations in Patients with vWD have a mucocuta- tion of platelets recruited to the site of pyruvate kinase. Review. Hum Mutat neous pattern of bleeding, with easy injury culminating in the formation of a 1996;7:1–6. haemostatically effective platelet plug. 14 van Wijk R, Rijksen G, van Solinge WW. bruising associated with trauma and may Molecular characterisation of pyruvate report spontaneous bruising. Epistaxes The high molecular weight multimer kinase deficiency – concerns about the and menorrhagia are common features. forms of vWF are required for these description of mutant PKLR alleles. Br J Prolonged bleeding following haemostatic platelet interactions. Haematol 2007;136:167–9. challenges, including minor cuts, dental vWF also functions as the carrier pro- 15 Kutlar A. Sickle cell disease: a multigenic tein for clotting factor VIII (FVIII), pro- perspective of a single gene disorder. extractions and surgical procedures, is Review. Haemoglobin 2007;31:209–24. also typical. tecting it from proteolytic degradation in 16 Vichinsky E. New therapies in sickle cell vWF is synthesised predominantly in the circulation and transporting it to disease. Review. Lancet 2002;360:629–31. blood vessel endothelial cells but is also sites of vascular injury to enable it to 17 Williamson D. The unstable produced by bone marrow megakary- carry out its crucial role in coagulation. haemoglobins. Review. Blood Rev Qualitative and quantitative defects in 1993;7:146–63. ocytes and incorporated into platelet alpha granules.2 The vWF gene is located vWF result in impairment of the haemo-

(b)

Platelet

GPIb

(a) (a) GPIIbIIIa

Fig 1. Role of von Willebrand factor (vWF) in haemostasis: (a) binding of vWF multimers to subendothelial collagen with capture of platelets, initially by binding to the glycoprotein (GP) Ib receptor and then to the GPIIbIIIa receptor; (b) aggregation of platelets by interbridging of vWF multimers between GPIIbIIIa receptors. Each vWF molecule can bind a factor VIII molecule (not shown) which is released at the site of haemostasis activation by the action of thrombin.

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