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Red cells II: acquired anaemias and polycythaemia
HAEMATOLOGY
H aem atology
Red cells II: acquired anaemias and polycythaemia Drew Provan, David Weatherall
• Iron deficiency affects 30% of the world’s population. • Iron metabolism is tightly regulated, with both gut transport and storage being coordinated. • Hereditary haemochromatosis due to mutations in the HFE gene leads to increased absorption of iron and multiple end-organ damage. • Myelodysplastic disorders are acquired clonal stem-cell disorders that cause ineffective erythropoiesis. • Aplastic anaemia is caused by an intrinsic defect of haemopoietic stem cells; both inherited and acquired forms occur. • Primary polycythaemia is a myeloproliferative disorder, a non-malignant stem-cell disease. This review focuses on the acquired anaemias, a large and diverse group of disorders (figure 1), ranging from frank blood loss to more subtle processes leading to premature senescence of red cells through various mechanisms. An acquired anaemia should be suspected in any patient who has previously normal blood counts, no family history of blood disorders, sudden onset of anaemia after infection or administration of a new drug, underlying systemic disease, onset of anaemia in later life, systemic symptoms, lymphadenopathy, masses, or altered bowel habit. Anaemia in these settings suggests an acquired disorder rather than an inherited anaemia, although some inherited disorders of red cells may present for the first time in adult life.
proteins facilitate iron acquisition. In human beings, three major proteins are involved in transport and storage of iron: plasma transferrin, transferrin receptor, and ferritin, the main storage form of iron. The average western daily diet contains 15 mg iron, of which 3 mg is taken up by the duodenal cells and 1 mg finally enters the plasma (panel 1). The proteins that bring about uptake of iron from the gut include the HFE gene product and NRAMP-2 (natural resistance-associated macrophage protein; also termed divalent-cation transporter [DCT] 1), which transport iron across the plasma membranes of the small-intestinal brush-border cells.3 The capacity of NRAMP-2 to transport iron Panel 1: iron function, acquisition, and homoeostasis
Anaemia due to iron deficiency Iron function, acquisition, and homoeostasis Iron deficiency is the commonest cause of anaemia (figure 2), affecting some 30% of the world population and accounting for up to 500 million cases worldwide.1 The prevalence is higher in less developed than in more developed societies (51% vs 8%). There have been few major advances in the clinical management of iron deficiency, and we have concentrated our review on recent developments that have provided insight into the understanding of iron homoeostasis, including control of iron uptake by the gut and coordination of the synthesis of storage and transport protein receptors. The investigation and treatment of iron deficiency are described elsewhere.2 Iron is essential for carriage of oxygen by haemoglobin and myoglobin, for oxidative metabolism, and for normal cellular growth. Although as a metal iron is abundant, most is in the highly insoluble ferric (Fe3+) form with low bioavailability. Because it is reactive and toxic, iron in the body is bound to various proteins for transport and storage. Lower eukaryotic organisms have proteins, termed siderophores, with high affinity for ferric iron; these Lancet 2000; 355: 1260–68 Department of Haematology, Southampton University Hospitals NHS Trust, Southampton SO16 6UY, UK (D Provan FRCP) and Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK (Prof D Weatherall FRS) Correspondence to: Dr Drew Provan (e-mail: [email protected])
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A normal western diet provides 15 mg iron per day but only 1 mg enters the circulation.
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The male adult has a mobile storage iron pool of no more than 1 g.
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Daily blood loss of 10–20 mL (containing 5–10 mg iron) will result in negative iron balance and depletion of storage iron fairly quickly.
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In iron deficiency the gut can increase its absorption of iron, but the maximum amount that can be absorbed daily is only 3–4 mg.
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The classic features of iron deficiency (decreased haemoglobin and hypochromic microcytic red cells), occur late in iron deficiency. In the early (latent) stages there is gradual depletion of storage iron, after which iron-deficient erythropoiesis occurs. At this stage the haemoglobin concentration, mean corpuscular volume, and mean corpuscular haemoglobin may still be within the normal range. Finally, with continuing negative iron balance, anaemia develops and microcytic hypochromic red cells appear in the peripheral blood.
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No specific excretion mechanism exists for iron: homoeostasis is controlled at the level of iron absorption by the gut.
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A male adult loses about 1 mg iron daily through cells shed from the gut and skin, in addition to iron lost in sweat and urine.
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Menstruating women have higher overall daily losses, due to the 30 mL (15 mg iron) or more lost monthly through menstrual bleeding.
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Acute blood loss Systemic disorders Anaemia of chronic disease Renal failure Liver failure Endocrine dysfunction
Deficiency disorders Iron Vitamin B12 Folate
Acquired progressive marrow disorders Myelodysplastic syndromes Paroxysmal nocturnal haemoglobinuria
Marrow infiltrations/fibrosis Metastatic cancer Leukaemia Lymphoma Storage disorders (eg, Gaucher's disease) Myelofibrosis
Physical destruction Burns Prosthetic heart valves Hypersplenism Haemolytic uraemic syndrome 'March' haemoglobinuria Venoms Oxidative damage eg, chlorates
Aplasia Idiopathic (50% cases) Drugs (eg, cytotoxics or idiosyncratic) Viral (eg, hepatitis, HIV)
Immune red-cell destruction Drug-induced Infection Autoimmune Primary (idiopathic) Secondary to lymphoid malignant disorders (eg, CLL, NHL) Connective-tissue disorders (eg, SLE) Infections (eg, mycoplasma, glandular fever) Drugs (eg, -methyldopa) Alloimmune
Rhesus haemolytic disease of newborn Haemolytic transfusion reaction
Figure 1: Causes of acquired anaemia CLL=chronic lymphocytic leukaemia; NHL=non-Hodgkin lymphoma; SLE=systemic lupus erythematosus.
depends on the body’s iron requirements at the time when the intestinal cell matures. Transferrin, the siderophore equivalent of higher organisms, is a metal-binding protein that carries iron in plasma, and cells that require iron express a receptor for transferrin on the cell surface.4 Transferrin, containing two bound iron atoms, attaches to its receptor. The receptor-transferrin complex is internalised, but not degraded, by the cell. After unloading the iron atoms, the complex returns to the cell surface and dissociates, leaving transferrin free to pick up further iron atoms. In iron deficiency and other disorders with increased erythropoiesis, such as haemolytic anaemias and polycythaemia vera, the number of transferrin receptors measurable in serum increases.5 HFE gene product, iron uptake, and hereditary haemochromatosis Hereditary haemochromatosis is characterised by progessive iron accumulation in various organs, including the heart and liver, and a predisposition to hepatocellular carcinoma in 25% of cases. Inheritance is autosomal recessive with a carrier rate of one in ten and a rate of affected homozygotes of one in 400. Homozygous individuals may develop skin pigmentation, hepatic dysfunction, hypothyroidism, hypoparathyroidism, adrenal insufficiency, cardiomyopathy, and chondrocalcinosis; heterozygous carriers do not develop these features. Patients present at age 40–60 years,
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typically with arthritis, features of gonadal failure, and diabetes mellitus. Laboratory tests show high serum ferritin concentrations (>1000 g/L in many cases), and liver biopsy confirms iron overloading. The underlying defect is a pronounced increase in intestinal iron absorption through upregulation of the duodenal metal transporter gene caused by mutation of the HFE gene.6 HFE is located on the short arm of chromosome 6 close to the HLA locus.7 Its product is widely expressed in most body tissues. 80–90% of patients
Figure 2: Iron-deficiency anaemia H=hypochromic red cell; p=pencil cell; T=target cell; M=microcytic cell. 500.
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with hereditary haemochromatosis are homozygous for a point mutation (G➝A at nucleotide 845) in HFE, which results in a cytosine to tyrosine replacement at aminoacid 282 (C282Y). This mutation provides a useful test for suspected homozygotes and for screening of family members. The HFE product binds to the transferrin receptor,8 although the mechanism through which these two proteins modulate iron absorption by the gut is poorly understood. In hereditary haemochromatosis, mutation of HFE leads to pronounced upregulation of NRAMP-2, with mRNA concentrations ten to 100 times higher than normal, leading to dysregulation of iron homoeostasis and progressive accumulation of iron in the heart, liver, and other tissues. Treatment involves regular removal of around 450 mL (1 unit) blood twice weekly for about 2 years to decrease the total body iron load, aiming to maintain the ferritin concentration in the lower end of the normal range by maintenance bleeding of 3 or 4 units per year. Coordination of iron transport and storage proteins In human beings, iron balance is mediated by ironregulatory proteins 1 and 2, which serve to coordinate the expression of ferritin, transferrin receptor, and NRAMP2. When iron is abundant, ferritin concentrations rise and transferrin-receptor concentrations decrease. Conversely, when iron is in short supply, ferritin concentrations fall and transferrin-receptor concentrations are upregulated. Control is exerted at the translational stage—ie, the ironregulatory proteins act on the mRNA molecules of ferritin and the transferrin receptor and either increase or suppress their translation into protein. The upstream (5 untranslated) region of ferritin mRNA contains a stem-loop structure, termed the ironresponsive element, which is recognised by ironregulatory protein 1. In the absence of iron, ironregulatory protein 1 represses ferritin synthesis9 through binding to ferritin mRNA and preventing translation of the ferritin message.10 Iron-regulatory protein 1 also controls the expression of the transferrin-receptor gene, located at 3q26.2-qter, close to the gene encoding transferrin. The downstream 3 untranslated region of the transferrin-receptor gene contains five stem-loop structures which are similar to the ferritin iron-responsive element.11 These conserved regions of the transferrinreceptor mRNA bind the same protein as the ironresponsive element of ferritin. In the absence of iron, ironregulatory protein 1 binds to transferrin-receptor mRNA, resulting in stabilisation of the mRNA and increased production of transferrin receptor. The converse occurs when iron is abundant: iron-regulatory protein 1 cannot bind to ferritin and transferrin-receptor mRNAs; therefore there is translation of ferritin and degradation of the unstable transferrin-receptor mRNA (figure 3). Assessment of iron status Several characteristics can be used to assess body iron status, including haemoglobin concentration, serum ferritin, serum iron and transferrin (as total iron-binding capacity); the bone-marrow aspirate stained for iron; and the soluble transferrin-receptor assay. The bone-marrow aspirate used to be the gold-standard method, but it has been largely superseded by the soluble transferrinreceptor assay. In simple iron deficiency, a low serum ferritin concentration confirms the diagnosis. However, serum concentrations of ferritin, an acute-phase protein, may be
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within or even above the normal range in inflammatory, malignant, or liver disease. Anaemia of chronic disease, found in patients with chronic inflammatory or infectious diseases, is a complex process involving inflammatory cytokines,12 a blunted marrow response to erythropoietin, reduced red-cell lifespan, and impaired iron reutilisation.13 In typical uncomplicated iron-deficiency anaemia, the mean corpuscular volume (MCV) and haemoglobin, serum ferritin, and serum iron concentrations are low with higher than normal total iron-binding capacity. However, if a patient with anaemia of chronic disease is also iron deficient, the features used for the diagnosis of iron deficiency are altered and the diagnosis is difficult, in many cases requiring a bone-marrow aspirate stained for iron. In iron-deficiency anaemia the number of transferrin receptors is upregulated and the soluble transferrinreceptor assay is now available to help distinguish iron deficiency from anaemia of chronic disease in cases for which the results of standard tests seem to conflict,5 thereby obviating the need for bone-marrow assessment. Since iron deficiency is a symptom rather than a diagnosis per se, the clinician must find out the underlying cause. In western societies, the commonest causes are gastrointestinal or menstrual blood loss. The other causes are well described in most major medical textbooks.14
Macrocytic anaemia caused by deficiency of vitamin B12 or folate Features of vitamin B12 and folate deficiency anaemias Vitamin B12 and folate are closely involved in DNA synthesis through methylation of deoxyuridine monophosphate to deoxythymidine monophosphate. IRP–1 +4Fe–4S High iron
Low iron –4Fe–4S
Cytoplasmic aconitase
IRE-binding protein
Translation inhibited
AAAA 3'
Ferritin coding
5' IRE
mRNA stabilised
5'
AAAA 3'
TfR coding IREs (5)
mRNA stabilised 5'
AAAA 3'
NRAMP-2 coding IRE
Figure 3: Coordinated expression of ferritin and transferrin receptor concentrations When intracellular iron concentration is low, iron-regulatory protein 1 (IRP1) binds to the mRNA iron-responsive element (IRE) stem-loop structure, inhibiting ferritin mRNA translation, but allowing increased expression of transferrin receptor through transferrin-receptor mRNA stabilisation. When iron concentrations are high, IRP-1 no longer binds to the IRE stem-loops; ferritin translation takes place but transferrin-receptor mRNA is degraded with a fall in transferrin-receptor protein. Reproduced with permission from Pippard MJ, Hoffbrand AV. Postgraduate haematology, 4th edn. In: Hoffrand AV, Mitchell SM, Tuddenham EGD, eds. Oxford: Butterworth Heinemann, 1999.
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Deficiency of either vitamin produces similar clinical and laboratory features.15 The symptoms are mainly those of chronic anaemia. However, deficiency of vitamin B12 is associated with neurological disturbance including paraesthesiae, loss of vibration sense, and, in extreme cases, myelin degeneration in the posterolateral columns resulting in subacute combined degeneration of the cord.16 Neurological complications can occur in the absence of anaemia. Thrombophilia has been linked with deficiency of vitamin B12 or folate through the hyperhomocysteinaemia that occurs when either vitamin is deficient. The homocysteine pathway involves enzymes that require vitamin B12, folate, and pyridoxine as cofactors. In nutritional deficiencies, frank deficiency of vitamin B12 or folate will lead to megaloblastic anaemia, whereas milder deficiencies are associated with a predisposition to thrombosis. In addition, hyperhomocysteinaemia predisposes to neural-tube defects. Molloy and colleagues17 found that 5–15% of normal western populations are homozygous for a point mutation (C667T) leading to partial deficiency of the key folate-metabolising enzyme, 5,10-methyltetrahydrofolate reductase, also associated with an increased rate of neuraltube defects and raised homocysteine concentrations. Deficiency of either vitamin B12 or folate typically presents as a macrocytic anaemia (MCV >110 fL in many
Figure 4: Megaloblastic anaemia HN=hypersegmented neutrophil; RBC=red blood cell; H-JB=Howell-Jolly body. 500.
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Panel 2: Causes of vitamin B12 and folate deficiency Causes of vitamin B12 deficiency Pernicious anaemia Nutritional (eg, vegan diet) Blind loops, bacterial overgrowth Ileal disease Gastrectomy Fish tapeworm (Diphyllobothrium latum) Causes of folate deficiency Dietary (eg, elderly people, alcoholic patients) Malabsorption (eg, coeliac disease, Crohn’s disease) Drugs (eg, anticonvulsants) Folate antagonists (eg, methotrexate, trimethoprim) Excessive loss/use (eg, pregnancy, haemolysis)
cases), although the MCV may be lower if there is coexisting iron deficiency, thalassaemia trait, inflammatory disease, or red-cell fragmentation.18 The blood film (figure 4) shows features of dyserythropoiesis, including oval macrocytosis, red-cell basophilic stippling, Howell-Jolly bodies, occasional circulating megaloblasts, and hypersegmented neutrophils. The white-cell and platelet counts may be low. Bone-marrow examination (figure 4), although seldom necessary now since assays for vitamin B12 are readily available, shows megaloblastic change with pronounced erythroid hyperplasia and predominance of early erythroid precursors, open atypical nuclear chromatin patterns, mitotic figures, and ‘giant’ metamyelocytes. Bone-marrow storage iron and serum ferritin are increased in most cases,19 the serum concentration of vitamin B12 is low, and serum and redcell folate concentrations are generally normal. Lactate dehydrogenase concentrations are high owing to ineffective erythropoiesis and early destruction of red cells.20 In western societies pernicious anaemia is the commonest cause of vitamin B12 deficiency (panel 2),21 with a rate of one in 10 000 in northern Europeans. Pernicious anaemia is due to autoimmune gastric atrophy and loss of intrinsic-factor production required for normal absorption of vitamin B12. Investigation and management Blood concentrations of both vitamins can be measured by ELISA methods.22 Vitamin B12 is measured as serum vitamin B12; folate may be measured as serum and redcell folate, the latter providing an estimate of body folate stores. Additional investigations in vitamin B12 deficiency include the measurement of serum intrinsic factor and parietal-cell antibodies, upper gastrointestinal endoscopy with duodenal biopsy, small-bowel barium studies, and other tests for malabsorption. Malabsorption of vitamin B12 can be tested by a urinary excretion method, such as the Schilling test,23 or whole-body B12 counting.24 Standard therapy consisting of five to six intramuscular injections of hydroxocobalamin (1 mg each) over a 3-week period corrects the haematological abnormalities in vitamin B12 deficiency and replaces stores. Patients are then given 1 mg intramuscularly every 3 months for life. Administration of folic acid in a patient with vitamin B12 deficiency may induce a haematological response but will worsen any neurological symptoms, and can actually precipitate subacute combined degeneration of the cord. In folate deficiency the serum and red-cell folate concentrations are low. Parietal-cell and intrinsic-factor autoantibodies are rarely present. Treatment consists of
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An acquired cause for haemolysis is likely if the patient presents for the first time in adult life, if previous blood counts have been normal, if there is no family history of haemolytic anaemia, or if the patient has recently been systemically unwell or has begun treatment with a new drug. There are many causes of haemolytic anaemia. Although specific diagnostic features may be present, there are features shared with many forms of haemolysis including: blood-film abnormalities such as polychromasia, spherocytosis, red-cell fragments, reticulocytosis, raised concentrations of serum bilirubin (unconjugated), urinary urobilinogen, and lactate dehydrogenase, and low or absent serum haptoglobins. Red-cell destruction can take place in the reticuloendothelial system (extravascular haemolysis) or in the circulation (intravascular haemolysis); the latter is associated with haemoglobinaemia, haemoglobinuria, haemosiderinuria, and methaemalbuminaemia, and follows, for example, ABO-mismatched transfusion. Extravascular haemolysis occurs in autoimmune haemolytic anaemias, rhesus-incompatible transfusion, and many chronic haemolytic disorders. The haemolysis can be immune or non-immune in origin. The direct antiglobulin test is positive in many cases with immune causes; positive reactions with specific reagents such as anti-IgG and anti-C3 suggest the presence of “warm” (IgG) or “cold” (IgM predominantly) antibodies, respectively. Investigation should also aim to find out whether an underlying disorder is causing the haemolysis or whether it is idiopathic. Underlying lymphoproliferative disorders such as chronic lymphocytic leukaemia, sepsis, renal or liver disease, and recent blood transfusion should be sought. Figure 5: Autoimmune haemolytic anaemia (warm, IgG antibody) RBC=red blood cell; reticulocyte=polychromatic (blue) RBC.
folic acid 5 mg daily for 4 months to replace stores. If there is an underlying cause such as coeliac disease, it should be treated. Folate prophylaxis Women planning pregnancy are advised to take 400 g folic acid daily before conception and until 12 weeks of pregnancy to prevent neural-tube defects (5 mg/day for women with a previous affected pregnancy).25 Folate fortification of cereal grains at 1·4 mg/kg has been made mandatory in the USA as an additional method of improving the folate status of the population. Prophylactic folate is also recommended in other states of increased demand such as long-term haemodialysis and chronic haemolytic disorders.26
Haemolytic anaemias due to reduced red-cell lifespan Acquired haemolytic disorders Haemolysis describes any situation in which the red-cell lifespan is less than 120 days. Anaemia results if there is failure of a compensatory marrow response. The predominant site of red-cell destruction is the red pulp of the spleen but other reticuloendothelial tissues are also involved.
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Autoimmune haemolytic anaemia Autoimmune haemolytic anaemia involves the production of red-cell autoantibodies, which may be warm or cold. Red cells react with autoantibody, with or without complement, causing their premature destruction by the reticuloendothelial system. The spleen and marrow ingest antibody-coated red cells, but in cases of incomplete phagocytosis spherical red cells are released into the peripheral circulation as spherocytes. Most warm autoimmune haemolytic anaemias are idiopathic, but this disorder may also be found in patients with underlying lymphoid malignant disorders or infections or in patients receiving certain classes of drugs.27,28 Presentation can be at any age but is commoner in adults. A full blood count shows a low concentration of haemoglobin, with spherocytes in the blood film (figure 5). The MCV is high in many cases, owing to the presence of reticulocytosis. Serological assays confirm the presence of IgG on the red-cell surface (by the direct antiglobulin test). Prednisolone 1 mg/kg daily for 1–2 weeks is the standard treatment; after this time the prednisolone dose is tailed off. The haemoglobin concentration rises and the reticulocyte count falls towards normal during this period. In cases of relapse other methods of immunosuppression may be used, including intravenous immunoglobulin. Splenectomy may be beneficial in some patients who do not respond to standard treatment. Cold autoimmune haemolytic anaemia occurs after infections (eg, Mycoplasma pneumoniae infection, glandular
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fever, or cytomegalovirus infection), and involves the production of polyclonal IgM autoantibodies. In elderly patients cold haemagglutinin disease is the result of a monoclonal IgM autoantibody. Investigations will confirm the presence of complement on the red-cell surface. Drug-induced haemolysis Drugs can induce haemolysis through various mechanisms,29 including: a hapten mechanism, in which the drug interacts with a red-cell component generating antigens that stimulate antibody production (eg, penicillins); autoantibody-mediated haemolysis, in which a warm autoantibody is generated (eg, -methyldopa); or an innocent-bystander mechanism in which the drug forms an immune complex with antibody that then attaches to the red cells leading to complement fixation and red-cell destruction (eg, quinine and quinidine). In addition, drugs can shorten the red-cell lifespan through interference with membrane lipids or through oxidation of haemoglobin. Non-immune red-cell destruction Physical agents can induce red-cell destruction through burns, damage on prosthetic heart valves, hypersplenism, haemolytic uraemic syndrome/thrombotic thrombocytopenic purpura, and march haemoglobinuria (in which red cells are destroyed in the soles of the feet of runners or soldiers).30
Anaemia due to clonal stem-cell disorders Myelodysplastic syndromes These syndromes are a morphologically and clinically heterogeneous group of acquired clonal bone-marrow stem-cell disorders predominantly affecting elderly people, although they may occur in younger adults and rarely in children.31 Myelodysplastic syndromes are characterised by ineffective bone-marrow haemopoiesis; dysplastic and defective blood cells are destroyed by the marrow before they ever enter the peripheral circulation, with resultant peripheral-blood cytopenias. The syndrome can be primary, arising de novo, or secondary to the administration of alkylating drugs including melphalan, chlorambucil, cyclophosphamide, and busulphan. Oncogene mutations have been reported in myelodysplastic syndromes; for example ras mutations have been detected in 9–40% cases and p53 mutations in 5–10%, although the role played by such mutations is unknown. The incidence of primary myelodysplastic syndromes increases with age—the annual incidence is 4·9 per 100 000 in the 50–70-year age-group and 22·8 per 100 000 in people older than 70. Although myelodysplastic syndromes were previously termed preleukaemic syndromes, many patients survive for many years with no evidence of development of leukaemia. Myelodysplastic syndromes are diagnosed by chance in about 20% of patients, when they have full blood counts done for other reasons. However, most patients present with symptoms reflecting the underlying peripheral-blood cytopenias—namely, anaemia, infection (caused by neutropenia or dysfunctional neutrophils) and bruising or bleeding (thrombocytopenia or dysfunctional platelets). The diagnosis is based on the blood count in conjunction with characteristic abnormalities of red and white blood cells and megakaryocytes in the bone marrow. The French-American-British classification32
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divides myelodysplastic syndromes into five subtypes based on bone-marrow morphology, immature (blast) cell count in blood and marrow, presence of iron-laden ring sideroblasts, and other cytological features. The subtypes are: refractory anaemia, so-called because these marrow disorders do not respond to iron or other haematinic replacement therapy; refractory anaemia with ring sideroblasts; refractory anaemia with excess blasts; refractory anaemia with excess blasts in transformation; and chronic myelomonocytic leukaemia. Refractory anaemia and refractory anaemia with ring sideroblasts have a good prognosis with median survival of 50 months. Refractory anaemia with excess blasts in transformation has a much poorer prognosis and median survival of only 6 months, largely due to the fact that this form, and refractory anaemia with excess blasts, are associated with a much higher leukaemic blast-cell count in the marrow and have a greater propensity to progress to frank acute myeloid leukaemia. Anaemia with a reduction in white-blood-cell and platelet counts is common. The blood film may show redcell abnormalities including variation in size and shape of red cells, macrocytosis, and basophilic stippling. Neutrophils have fewer cytoplasmic granules than normal (hypogranular) and have characteristic abnormalities of the nucleus. Despite the peripheral-blood cytopenias typical of myelodysplastic syndromes, the bone marrow is hypercellular in many cases, reflecting the ineffective haemopoiesis associated with the disorders. Acquired (ie, non-constitutional) chromosomal abnormalities are common in myelodysplastic syndromes, and analysis of bone-marrow chromosomes provides useful prognostic information. For example, patients with normal karyotypes or loss of the long arm of chromosome 5 (5q abnormality33) have a far better prognosis than those with loss of chromosome 7, deletions of 7q, or more complex karyotypes. Acquired chromosomal abnormalities are present in at least 50% of patients with primary myelodysplastic syndromes, and are more common in poor-prognosis (>60% patients with refractory anaemia with excess blasts in transformation) than in good-prognosis myelodysplastic syndromes (<30% in refractory anaemia). Chromosome abnormalities associated with poor prognosis include loss of chromosome 7 (-7), deletion of the long arm of chromosome 7 (del7q) or short arm of chromosome 17 (del17p), complex abnormalities involving multiple chromosomes, and evolving chromosome abnormalities particularly if the chromosomes were previously normal. Most patients with myelodysplastic syndromes are elderly and the management is essentially supportive. Red-cell transfusions are given when patients have symptoms caused by their anaemia. Thrombocytopenic bleeding can be helped by regular platelet transfusion. Infection is the main cause of morbidity and mortality in patients with myelodysplastic syndromes. Patients who develop infection should be treated promptly with antibiotics. Bone-marrow transplantation is an option for younger patients (eg, those younger than 50 years).34 Paroxysmal nocturnal haemoglobinuria Paroxysmal nocturnal haemoglobinuria is a rare acquired clonal disorder of the haemopoietic stem cell, characterised by chronic intravascular haemolysis, thrombotic tendency, and variable degrees of bone-
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marrow failure. The underlying defect is somatic mutation of the X-linked gene for phosphatidylinositol glycan protein A; this protein is required for production of glycerylphosphatidylinositol, which protects the red-cell membrane against complement-mediated damage. Other proteins implicated in paroxysmal nocturnal haemoglobinuria include decay-accelerating factor (or CD55) and CD59; when these proteins are absent, complement-mediated red-cell lysis occurs. The disorder affects young people but may be diagnosed in childhood or later life. The patient complains of passing dark urine, usually on waking. The haemoglobin concentration is low, and the MCV is high (caused by reticulocytosis, bone-marrow failure, or both). The white-cell and platelet counts are low in many cases. Other features of haemolysis are commonly present, including raised serum lactate dehydrogenase and bilirubin, with absence of haptoglobins. Chronic haemoglobinuria and haemosiderinuria lead to high urinary iron loss and ultimately iron deficiency. Together with these features, a positive Ham’s acidified serum test supports the diagnosis of paroxysmal nocturnal haemoglobinuria. Flow-cytometric analysis of red cells with anti-CD55 and anti-CD59 has replaced the Ham’s test. The increased risk of thrombosis, which may affect mesenteric, hepatic (Budd-Chiari syndrome), or other veins, is probably due to complement-mediated activation of platelets deficient in CD55 and CD59. The median survival is 10 years or longer, and the disorder has a close association with aplastic anaemia. Serious thrombosis occurs in 20% of patients, and a small proportion (5%) show progression to acute leukaemia. Treatment is essentially supportive and includes prompt treatment of infection, red-cell transfusion, and folic acid and iron supplementation. Thrombotic complications require vigorous treatment followed by life-long oral anticoagulation. In patients with severe bone-marrow failure, treatment options include immunosuppression with antilymphocyte globulin or cyclosporin, or siblingdonor bone-marrow transplantation. Bone-marrow transplantation from a matched unrelated donor may be an option for the treatment of severe paroxysmal nocturnal haemoglobinuria in patients younger than 25 years.35,36 Aplastic anaemia Aplastic anaemia, one form of bone-marrow-failure syndrome, describes the presence of peripheral-blood pancytopenia with a grossly hypocellular bone marrow, in which there is replacement of normal marrow haemopoietic cells by fat cells.37 By definition, there are no cancerous, leukaemic or other abnormal cells in either blood or marrow. There is evidence that aplastic anaemia is caused by an intrinsic defect of the haemopoietic stem cells that results in variable degrees of stem-cell loss. For example, syngeneic (identical twin) bone-marrow transplantation, without the use of immunosuppression, results in cure in 50% of cases.38 Other mechanisms invoked in the pathogenesis of aplastic anaemia include abnormalities of the bone-marrow stromal microenvironment, immune suppression of the marrow, and growth-factor deficiency. Since patients show variable responses to treatment, and the disease evolution is diverse, aplastic anaemia probably represents a heterogeneous group of disorders, rather than a single disease entity.
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Aplastic anaemia can be hereditary (for example, Fanconi anaemia, Diamond-Blackfan syndrome) or acquired; the latter includes the inevitable aplasia that follows cytotoxic chemotherapy or radiotherapy; idiosyncratic (drugs); postviral (hepatitis, cytomegalovirus, Epstein-Barr virus); and immune-mediated (for example, systemic lupus erythematosus). Around two-thirds of cases of aplastic anaemia are idiopathic, with no obvious underlying cause. Idiopathic aplastic anaemia is rare, with an annual incidence of two to five cases per million population (although the disorder is ten times more common in Oriental populations). Some patients have few symptoms at presentation and others have non-specific features including tiredness, bleeding, and a propensity to infection caused by decreased haemoglobin, platelets, and white blood cells, respectively. The full blood count shows variable degrees of pancytopenia with lower than normal haemoglobin concentration, white-cell count, and platelet count and a blood film that lacks major pathological features. Reticulocytes (young red blood cells) are low in number or absent from peripheral blood. The bone-marrow aspirate and trephine biopsy show replacement of normal haemopoietic tissue by fat cells, and the few haemopoietic cells that remain are lymphocytes and plasma cells. The Ham’s acidified serum lysis test is negative. Idiopathic aplastic anaemia can be stable, progressive, or unstable.37 Patients with severe forms (neutrophils <0·5109/L, platelets <20109/L, reticulocytes <1%) have a one in ten chance of surviving 1 year from diagnosis with only supportive treatment. Patients in whom the pancytopenia is less pronounced fare somewhat better. The progressive group has pancytopenia that worsens with time. Finally, the unstable group may develop abnormal clonal marrow cell populations. For example, by means of laboratory tests such as the Ham’s test, paroxysmal nocturnal haemoglobinuria clones are found in 10–20% of patients with prolonged idiopathic aplastic anaemia, although only a minority develop florid paroxysmal nocturnal haemoglobinuria. Other abnormal clones include acute myeloid leukaemia and myelodysplastic syndromes. Management consists of supportive therapy and treatment aimed at restoring normal bone-marrow activity. Supportive therapy involves red-cell and platelet transfusions, prophylactic antibiotics and antifungals, and general hygiene measures. If patients are treated solely in this way, only 28% are alive at 2 years from diagnosis.39 Specific measures to restore marrow activity include the use of immunosuppression, such as antilymphocyte globulin, androgens (eg, oxymethalone), and haemopoietic growth factors such as granulocyte colonystimulating factor. Sibling-donor bone-marrow or stemcell transplantation, offering long-term survival of more than 65%, should be considered in patients younger than 50 years who have suitable donors. Over a period of 10 years up to 40% of patients who respond to immunosuppression may develop paroxysmal nocturnal haemoglobinuria, myelodysplasia, or acute myeloid leukaemia.40
Acquired polycythaemia Polycythaemias The polycythaemias are red-cell disorders associated with an increase in haemoglobin concentration, packed-cell
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Panel 3: Erythropoietin concentrations in secondary polycythaemias Appropriate erythropoietin increase Hypoxia Chronic lung disease Heavy smoking (carboxyhaemoglobin) High altitude Congenital cyanotic heart disease Gross obesity High-affinity haemoglobin (familial erythrocytosis) Inappropriate erythropoietin increase Renal disorders Hypernephroma Hydronephrosis Polycystic kidney disease Post-renal transplant Other Hepatoma Uterine leiomyoma Cerebellar haemangioblastoma
volume, and red-cell count, and are subdivided into absolute erythrocytosis (true increase in red-cell mass) and relative erythrocytosis (red-cell mass normal but plasma volume reduced). A red-cell mass greater than 25% above the predicted value indicates absolute polycythaemia. Three main groups are recognised: primary proliferative polycythaemia (also called polycythaemia rubra vera),41 secondary polycythaemia (due to appropriate or inappropriate increases in erythropoietin concentration), and apparent polycythaemia42 (older terms include relative, spurious, and Gaisbock’s syndrome). Only the first two groups have a true increase in red-cell mass. Primary polycythaemia Polycythaemia rubra vera is one of the myeloproliferative disorders, a group of non-malignant clonal bone-marrow stem-cell disorders that includes essential thrombocythaemia, idiopathic myelofibrosis, and chronic myeloid leukaemia. The underlying cause is unknown but is likely to be acquired genetic changes in the stem cell leading to disturbance of the normal cellular growth pattern. Cellculture studies have shown that the red-cell precursors are able to grow independently of erythropoietin and are more sensitive than normal to various growth factors including erythropoietin and thrombopoietin. Cellular proliferation is believed to be driven through protein phosphorylation, and the tumour suppressor gene, SHP-1, seems to be a suitable candidate in this process; the SHP-1 product interacts with several growth-factor receptors including that of erythropoietin. In addition, loss of the SHP-1 binding domain from the erythropoietin receptor results in one form of congenital polycythaemia.43 Polycythaemia rubra vera is a disorder of older people (median age 55–60 years) of both sexes; it may be a chance finding, or patients may present with vascular or other complications including digital ischaemia, cerebrovascular accident, transient ischaemic attack, myocardial infarction, bleeding, bruising, hyperuricaemia/gout, or itching. The full blood count shows increased haemoglobin concentration, packed-cell volume, and red-cell count; 70% of cases have high white-blood-cell and platelet counts. The MCV may be low owing to concomitant iron deficiency caused through, for example, gastrointestinal bleeding. The red-cell mass is higher than normal (>36
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mL/kg in men; >32 mL/kg in women). The bone marrow is hypercellular with increased red cells and megakaryocytes, though this feature is not diagnostic in isolation. Chromosome studies are not carried out routinely but previous studies have shown chromosomal abnormalities in 15% of patients, including trisomy 1, 8, and 9, and deletion of 13q and 20q. The packed-cell volume is decreased by removal of 1 unit of blood every alternate day until the packed-cell volume is within desired range, then every 6–8 weeks. Alternatively, the packed-cell volume can be controlled by use of oral chemotherapy, such as hydroxyurea, an inhibitor of ribonucleotide reductase. Busulphan is no longer the drug of choice because it induces secondary leukaemias in later life and pulmonary fibrosis. Phosphorus-32, reserved for use in elderly patients, may provide control of packed-cell volume for up to 18 months. Polycythaemia rubra vera may progress to myelofibrosis (30%) or acute myeloid leukaemia (5%) with a median survival of 10–16 years. Secondary polycythaemias These disorders are predominantly driven by hypoxia, resulting in a physiologically appropriate compensatory increase in erythropoietin production; a smaller proportion are caused by inappropriate erythropoietin production (panel 3). The complications are similar to those of polycythaemia rubra vera but there is no progression to myelofibrosis or acute leukaemia. Treatment aims to reduce the packed-cell volume to a reasonably safe value, but care should be taken in patients whose increase in red-cell mass is compensatory, since over-vigorous treatment may be detrimental. Apparent polycythaemia This term describes patients with increased haemoglobin concentration and packed-cell volume but normal red-cell mass. A reduction in plasma volume causes the erythrocytosis. Apparent polycythaemia is more common than polycythaemia rubra vera and typically affects middle-aged men who are obese and hypertensive, who smoke, and who have high alcohol consumption. In some cases apparent polycythaemia may be associated with diuretic therapy. Treatment is that of the underlying disorder. The patient should reduce weight and cigarette consumption. Venesection to a packed-cell volume of less than 50% may be required in patients with definite cardiovascular risks. References 1 Cook JD, Skikne BS, Baynes RD. Iron deficiency: the global perspective. Adv Exp Med Biol 1994; 356:219–28. 2 Provan D. Mechanisms and management of iron deficiency anaemia. Br J Haematol 1999; 105 (suppl 1):19–26. 3 Gunshin H, Mackenzie B, Berger UV, et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997; 388:482–88. 4 Seligman PA, Schleicher RB, Allen RH. Isolation and characterization of the transferrin receptor from human placenta. J Biol Chem 1979; 254:9943–46. 5 Kohgo Y, Niitsu Y, Nishisato T, et al. Quantitation and characterization of serum transferrin receptor in patients with anemias and polycythemias. Jpn J Med 1988; 27:67–40. 6 Zoller H, Pietroangelo A Vogel W, Weiss G. Duodenal metal transporter (DMT-1, NRAMP-2) expression in patients with hereditary haeochromatosis. Lancet 1999; 353:2120–23. 7 Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996; 13:399–408.
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HAEMATOLOGY 8 Waheed A, Parkkila S, Saarnio J, et al. Association of HFE protein with transferrin receptor in crypt enterocytes of human duodenum. Proc Natl Acad Sci USA 1999; 96:1579–84. 9 Gray NK, Hentze MW. Iron regulatowry protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs. EMBO J 1994; 13:3882–91. 10 Kaptain S, Downey WE, Tang C, et al. A regulated RNA binding protein also possesses aconitase activity. Proc Natl Acad Sci USA 1991; 88:10109–13. 11 Mullner EW, Kuhn LC. A stem-loop in the 3’ untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm. Cell 1988; 53:815–25. 12 Means RT, Krantz SB. Progress in understanding the pathogenesis of the anemia in chronic disease. Blood 1992; 80:1639–47. 13 Baer AN, Dessypris EN, Krantz SB. The pathogenesis of anemia in rheumatoid arthritis: a clinical and laboratory analysis. Semin Arthritis Rheum 1990; 19:209–23. 14 Pippard MJ, Hoffbrand AV. Iron. In: Hoffbrand AV, Lewis SM, Tuddenham EGD, eds. Postgraduate haematology, 4th edn. Oxford: Butterworth-Heinemann, 1999: 23–46. 15 Wickramasinghe SN. Morphology, biology and biochemistry of cobalamin-and folate-deficient bone marrow cells. Baillieres Clin Haematol 1995; 8:441–59. 16 Savage DG, Lindenbaum J. Neurological complications of acquired cobalamin deficiency: clinical aspects. Baillieres Clin Haematol 1995; 8: 657–78. 17 Molloy AM, Daly S, Mills JL, et al. Thermolabile variant of 5,10methylenetetrahydrofolate reductase associated with low red-cell folates: implications for folate intake recommendations. Lancet 1997; 349:1591–93. 18 Spivak JL. Masked megaloblastic anemia. Arch Intern Med 1982; 142: 2111–14. 19 Hussein S, Laulicht M, Hoffbrand AV. Serum ferritin in megaloblastic anaemia. Scand J Haematol 1978; 20:241–45. 20 Winston RM, Warburton FG, Stott A. Enzymatic diagnosis of megaloblastic anaemia. Br J Haematol 1970; 19:587–92. 21 Toh BH, van Driel IR, Gleeson PA. Pernicious anemia. N Engl J Med 1997; 337:1441–48. 22 Chanarin I, Metz J. Diagnosis of cobalamin deficiency: the old and the new. Br J Haematol 1997; 97:695–700. 23 Fairbanks VF, Wahner HW, Phyliky RL. Tests for pernicious anemia: the Schilling test. Mayo Clin Proc 1983; 58:541–44. 24 Callender ST, Witts LJ, Warner GT, Oliver R. The use of a simple whole-body counter for haematological investigations. Br J Haematol 1966; 12:276–82. 25 Department of Health, Expert Advisory Group. Folic acid and the prevention of neural tube defect. London: Department of Health, 1992.
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26 Hoffbrand AV. Megaloblastic anaemia. In: Hoffbrand AV, Lewis SM, Tuddenham EGD, eds. Postgraduate haematology, 4th edn. Oxford: Butterworth-Heinemann, 1999: 47–67. 27 Dacie JV, ed. Autoimmune haemolytic anaemias. Edinburgh: Churchill Livingstone, 1992. 28 Petz LD. Drug-induced autoimmune hemolytic anemias. Transfus Med Rev 1993; 7:242–54. 29 Gordon-Smith EC, Hows JM, Luzzatto L. Acquired haemolytic anaemias. In: Hoffbrand AV, Lewis SM, Tuddenham EGD, eds. Postgraduate haematology, 4th edn. Oxford: Butterworth-Heinemann, 1999: 144–63. 30 Hallberg L, Magnusson B. The etiology of “sports anemia”: a physiological adaptation of the oxygen-dissociation curve of hemoglobin to an unphysiological exercise load. Acta Med Scand 1984; 216:147–8. 31 Passmore SJ, Hann IM, Stiller CA, et al. Pediatric myelodysplasia: a study of 68 children and a new prognostic scoring system. Blood 1995; 85:1742–50. 32 Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982; 51:189–99. 33 Boultwood J, Lewis S, Wainscoat JS. The 5q- syndrome. Blood 1994; 84:3253–60. 34 Anderson JE, Anasetti C, Appelbaum FR, et al. Unrelated donor marrow transplantation for myelodysplasia (MDS) and MDS-related acute myeloid leukaemia. Br J Haematol 1996; 93:59–67. 35 Rosse WF, Ware RE. The molecular basis of paroxysmal nocturnal hemoglobinuria. Blood 1995; 86:3277–86. 36 Socie G, Mary JY, de Gramont A, et al. Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. Lancet 1996; 348:573–77. 37 Gordon-Smith EC, Lewis SM. Aplastic anaemia and other types of bone marrow failure. In: Hoffbrand AV, Lewis SM, Tuddenham EGD, eds. Postgraduate haematology. 4th edn. Oxford: ButterworthHeinemann, 1999: 68–90. 38 Champlin RE, Feig SA, Sparkes RS, Galen RP. Bone marrow transplantation from identical twins in the treatment of aplastic anaemia: implication for the pathogenesis of the disease. Br J Haematol 1984; 56:455–63. 39 Camitta BM, Thomas ED, Nathan DG, et al. A prospective study of androgens and bone marrow transplantation for treatment of severe aplastic anemia. Blood 1979; 53:504–14. 40 de Planque MM, Kluin-Nelemans HC, van Krieken HJ, et al. Evolution of acquired severe aplastic anaemia to myelodysplasia and subsequent leukaemia in adults. Br J Haematol 1988; 70:55–62. 41 Berlin NI. Polycythemia vera. Semin Hematol 1997; 34:1–80. 42 Pearson TC. Apparent polycythaemia. Blood Rev 1991; 5:205–13. 43 Bench AJ, Huntly BJP, Nacheva EP, Champion KM, Green AR. Myeloproliferative disorders. In: Provan D, Gribben JG, eds. Molecular haematology. Oxford: Blackwell Science, 2000.
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H aem atology
Red cells II: acquired anaemias and polycythaemia Drew Provan, David Weatherall
• Iron deficiency affects 30% of the world’s population. • Iron metabolism is tightly regulated, with both gut transport and storage being coordinated. • Hereditary haemochromatosis due to mutations in the HFE gene leads to increased absorption of iron and multiple end-organ damage. • Myelodysplastic disorders are acquired clonal stem-cell disorders that cause ineffective erythropoiesis. • Aplastic anaemia is caused by an intrinsic defect of haemopoietic stem cells; both inherited and acquired forms occur. • Primary polycythaemia is a myeloproliferative disorder, a non-malignant stem-cell disease. This review focuses on the acquired anaemias, a large and diverse group of disorders (figure 1), ranging from frank blood loss to more subtle processes leading to premature senescence of red cells through various mechanisms. An acquired anaemia should be suspected in any patient who has previously normal blood counts, no family history of blood disorders, sudden onset of anaemia after infection or administration of a new drug, underlying systemic disease, onset of anaemia in later life, systemic symptoms, lymphadenopathy, masses, or altered bowel habit. Anaemia in these settings suggests an acquired disorder rather than an inherited anaemia, although some inherited disorders of red cells may present for the first time in adult life.
proteins facilitate iron acquisition. In human beings, three major proteins are involved in transport and storage of iron: plasma transferrin, transferrin receptor, and ferritin, the main storage form of iron. The average western daily diet contains 15 mg iron, of which 3 mg is taken up by the duodenal cells and 1 mg finally enters the plasma (panel 1). The proteins that bring about uptake of iron from the gut include the HFE gene product and NRAMP-2 (natural resistance-associated macrophage protein; also termed divalent-cation transporter [DCT] 1), which transport iron across the plasma membranes of the small-intestinal brush-border cells.3 The capacity of NRAMP-2 to transport iron Panel 1: iron function, acquisition, and homoeostasis
Anaemia due to iron deficiency Iron function, acquisition, and homoeostasis Iron deficiency is the commonest cause of anaemia (figure 2), affecting some 30% of the world population and accounting for up to 500 million cases worldwide.1 The prevalence is higher in less developed than in more developed societies (51% vs 8%). There have been few major advances in the clinical management of iron deficiency, and we have concentrated our review on recent developments that have provided insight into the understanding of iron homoeostasis, including control of iron uptake by the gut and coordination of the synthesis of storage and transport protein receptors. The investigation and treatment of iron deficiency are described elsewhere.2 Iron is essential for carriage of oxygen by haemoglobin and myoglobin, for oxidative metabolism, and for normal cellular growth. Although as a metal iron is abundant, most is in the highly insoluble ferric (Fe3+) form with low bioavailability. Because it is reactive and toxic, iron in the body is bound to various proteins for transport and storage. Lower eukaryotic organisms have proteins, termed siderophores, with high affinity for ferric iron; these Lancet 2000; 355: 1260–68 Department of Haematology, Southampton University Hospitals NHS Trust, Southampton SO16 6UY, UK (D Provan FRCP) and Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK (Prof D Weatherall FRS) Correspondence to: Dr Drew Provan (e-mail: [email protected])
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A normal western diet provides 15 mg iron per day but only 1 mg enters the circulation.
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The male adult has a mobile storage iron pool of no more than 1 g.
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Daily blood loss of 10–20 mL (containing 5–10 mg iron) will result in negative iron balance and depletion of storage iron fairly quickly.
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In iron deficiency the gut can increase its absorption of iron, but the maximum amount that can be absorbed daily is only 3–4 mg.
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The classic features of iron deficiency (decreased haemoglobin and hypochromic microcytic red cells), occur late in iron deficiency. In the early (latent) stages there is gradual depletion of storage iron, after which iron-deficient erythropoiesis occurs. At this stage the haemoglobin concentration, mean corpuscular volume, and mean corpuscular haemoglobin may still be within the normal range. Finally, with continuing negative iron balance, anaemia develops and microcytic hypochromic red cells appear in the peripheral blood.
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No specific excretion mechanism exists for iron: homoeostasis is controlled at the level of iron absorption by the gut.
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A male adult loses about 1 mg iron daily through cells shed from the gut and skin, in addition to iron lost in sweat and urine.
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Menstruating women have higher overall daily losses, due to the 30 mL (15 mg iron) or more lost monthly through menstrual bleeding.
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Acute blood loss Systemic disorders Anaemia of chronic disease Renal failure Liver failure Endocrine dysfunction
Deficiency disorders Iron Vitamin B12 Folate
Acquired progressive marrow disorders Myelodysplastic syndromes Paroxysmal nocturnal haemoglobinuria
Marrow infiltrations/fibrosis Metastatic cancer Leukaemia Lymphoma Storage disorders (eg, Gaucher's disease) Myelofibrosis
Physical destruction Burns Prosthetic heart valves Hypersplenism Haemolytic uraemic syndrome 'March' haemoglobinuria Venoms Oxidative damage eg, chlorates
Aplasia Idiopathic (50% cases) Drugs (eg, cytotoxics or idiosyncratic) Viral (eg, hepatitis, HIV)
Immune red-cell destruction Drug-induced Infection Autoimmune Primary (idiopathic) Secondary to lymphoid malignant disorders (eg, CLL, NHL) Connective-tissue disorders (eg, SLE) Infections (eg, mycoplasma, glandular fever) Drugs (eg, -methyldopa) Alloimmune
Rhesus haemolytic disease of newborn Haemolytic transfusion reaction
Figure 1: Causes of acquired anaemia CLL=chronic lymphocytic leukaemia; NHL=non-Hodgkin lymphoma; SLE=systemic lupus erythematosus.
depends on the body’s iron requirements at the time when the intestinal cell matures. Transferrin, the siderophore equivalent of higher organisms, is a metal-binding protein that carries iron in plasma, and cells that require iron express a receptor for transferrin on the cell surface.4 Transferrin, containing two bound iron atoms, attaches to its receptor. The receptor-transferrin complex is internalised, but not degraded, by the cell. After unloading the iron atoms, the complex returns to the cell surface and dissociates, leaving transferrin free to pick up further iron atoms. In iron deficiency and other disorders with increased erythropoiesis, such as haemolytic anaemias and polycythaemia vera, the number of transferrin receptors measurable in serum increases.5 HFE gene product, iron uptake, and hereditary haemochromatosis Hereditary haemochromatosis is characterised by progessive iron accumulation in various organs, including the heart and liver, and a predisposition to hepatocellular carcinoma in 25% of cases. Inheritance is autosomal recessive with a carrier rate of one in ten and a rate of affected homozygotes of one in 400. Homozygous individuals may develop skin pigmentation, hepatic dysfunction, hypothyroidism, hypoparathyroidism, adrenal insufficiency, cardiomyopathy, and chondrocalcinosis; heterozygous carriers do not develop these features. Patients present at age 40–60 years,
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typically with arthritis, features of gonadal failure, and diabetes mellitus. Laboratory tests show high serum ferritin concentrations (>1000 g/L in many cases), and liver biopsy confirms iron overloading. The underlying defect is a pronounced increase in intestinal iron absorption through upregulation of the duodenal metal transporter gene caused by mutation of the HFE gene.6 HFE is located on the short arm of chromosome 6 close to the HLA locus.7 Its product is widely expressed in most body tissues. 80–90% of patients
Figure 2: Iron-deficiency anaemia H=hypochromic red cell; p=pencil cell; T=target cell; M=microcytic cell. 500.
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with hereditary haemochromatosis are homozygous for a point mutation (G➝A at nucleotide 845) in HFE, which results in a cytosine to tyrosine replacement at aminoacid 282 (C282Y). This mutation provides a useful test for suspected homozygotes and for screening of family members. The HFE product binds to the transferrin receptor,8 although the mechanism through which these two proteins modulate iron absorption by the gut is poorly understood. In hereditary haemochromatosis, mutation of HFE leads to pronounced upregulation of NRAMP-2, with mRNA concentrations ten to 100 times higher than normal, leading to dysregulation of iron homoeostasis and progressive accumulation of iron in the heart, liver, and other tissues. Treatment involves regular removal of around 450 mL (1 unit) blood twice weekly for about 2 years to decrease the total body iron load, aiming to maintain the ferritin concentration in the lower end of the normal range by maintenance bleeding of 3 or 4 units per year. Coordination of iron transport and storage proteins In human beings, iron balance is mediated by ironregulatory proteins 1 and 2, which serve to coordinate the expression of ferritin, transferrin receptor, and NRAMP2. When iron is abundant, ferritin concentrations rise and transferrin-receptor concentrations decrease. Conversely, when iron is in short supply, ferritin concentrations fall and transferrin-receptor concentrations are upregulated. Control is exerted at the translational stage—ie, the ironregulatory proteins act on the mRNA molecules of ferritin and the transferrin receptor and either increase or suppress their translation into protein. The upstream (5 untranslated) region of ferritin mRNA contains a stem-loop structure, termed the ironresponsive element, which is recognised by ironregulatory protein 1. In the absence of iron, ironregulatory protein 1 represses ferritin synthesis9 through binding to ferritin mRNA and preventing translation of the ferritin message.10 Iron-regulatory protein 1 also controls the expression of the transferrin-receptor gene, located at 3q26.2-qter, close to the gene encoding transferrin. The downstream 3 untranslated region of the transferrin-receptor gene contains five stem-loop structures which are similar to the ferritin iron-responsive element.11 These conserved regions of the transferrinreceptor mRNA bind the same protein as the ironresponsive element of ferritin. In the absence of iron, ironregulatory protein 1 binds to transferrin-receptor mRNA, resulting in stabilisation of the mRNA and increased production of transferrin receptor. The converse occurs when iron is abundant: iron-regulatory protein 1 cannot bind to ferritin and transferrin-receptor mRNAs; therefore there is translation of ferritin and degradation of the unstable transferrin-receptor mRNA (figure 3). Assessment of iron status Several characteristics can be used to assess body iron status, including haemoglobin concentration, serum ferritin, serum iron and transferrin (as total iron-binding capacity); the bone-marrow aspirate stained for iron; and the soluble transferrin-receptor assay. The bone-marrow aspirate used to be the gold-standard method, but it has been largely superseded by the soluble transferrinreceptor assay. In simple iron deficiency, a low serum ferritin concentration confirms the diagnosis. However, serum concentrations of ferritin, an acute-phase protein, may be
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within or even above the normal range in inflammatory, malignant, or liver disease. Anaemia of chronic disease, found in patients with chronic inflammatory or infectious diseases, is a complex process involving inflammatory cytokines,12 a blunted marrow response to erythropoietin, reduced red-cell lifespan, and impaired iron reutilisation.13 In typical uncomplicated iron-deficiency anaemia, the mean corpuscular volume (MCV) and haemoglobin, serum ferritin, and serum iron concentrations are low with higher than normal total iron-binding capacity. However, if a patient with anaemia of chronic disease is also iron deficient, the features used for the diagnosis of iron deficiency are altered and the diagnosis is difficult, in many cases requiring a bone-marrow aspirate stained for iron. In iron-deficiency anaemia the number of transferrin receptors is upregulated and the soluble transferrinreceptor assay is now available to help distinguish iron deficiency from anaemia of chronic disease in cases for which the results of standard tests seem to conflict,5 thereby obviating the need for bone-marrow assessment. Since iron deficiency is a symptom rather than a diagnosis per se, the clinician must find out the underlying cause. In western societies, the commonest causes are gastrointestinal or menstrual blood loss. The other causes are well described in most major medical textbooks.14
Macrocytic anaemia caused by deficiency of vitamin B12 or folate Features of vitamin B12 and folate deficiency anaemias Vitamin B12 and folate are closely involved in DNA synthesis through methylation of deoxyuridine monophosphate to deoxythymidine monophosphate. IRP–1 +4Fe–4S High iron
Low iron –4Fe–4S
Cytoplasmic aconitase
IRE-binding protein
Translation inhibited
AAAA 3'
Ferritin coding
5' IRE
mRNA stabilised
5'
AAAA 3'
TfR coding IREs (5)
mRNA stabilised 5'
AAAA 3'
NRAMP-2 coding IRE
Figure 3: Coordinated expression of ferritin and transferrin receptor concentrations When intracellular iron concentration is low, iron-regulatory protein 1 (IRP1) binds to the mRNA iron-responsive element (IRE) stem-loop structure, inhibiting ferritin mRNA translation, but allowing increased expression of transferrin receptor through transferrin-receptor mRNA stabilisation. When iron concentrations are high, IRP-1 no longer binds to the IRE stem-loops; ferritin translation takes place but transferrin-receptor mRNA is degraded with a fall in transferrin-receptor protein. Reproduced with permission from Pippard MJ, Hoffbrand AV. Postgraduate haematology, 4th edn. In: Hoffrand AV, Mitchell SM, Tuddenham EGD, eds. Oxford: Butterworth Heinemann, 1999.
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Deficiency of either vitamin produces similar clinical and laboratory features.15 The symptoms are mainly those of chronic anaemia. However, deficiency of vitamin B12 is associated with neurological disturbance including paraesthesiae, loss of vibration sense, and, in extreme cases, myelin degeneration in the posterolateral columns resulting in subacute combined degeneration of the cord.16 Neurological complications can occur in the absence of anaemia. Thrombophilia has been linked with deficiency of vitamin B12 or folate through the hyperhomocysteinaemia that occurs when either vitamin is deficient. The homocysteine pathway involves enzymes that require vitamin B12, folate, and pyridoxine as cofactors. In nutritional deficiencies, frank deficiency of vitamin B12 or folate will lead to megaloblastic anaemia, whereas milder deficiencies are associated with a predisposition to thrombosis. In addition, hyperhomocysteinaemia predisposes to neural-tube defects. Molloy and colleagues17 found that 5–15% of normal western populations are homozygous for a point mutation (C667T) leading to partial deficiency of the key folate-metabolising enzyme, 5,10-methyltetrahydrofolate reductase, also associated with an increased rate of neuraltube defects and raised homocysteine concentrations. Deficiency of either vitamin B12 or folate typically presents as a macrocytic anaemia (MCV >110 fL in many
Figure 4: Megaloblastic anaemia HN=hypersegmented neutrophil; RBC=red blood cell; H-JB=Howell-Jolly body. 500.
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Panel 2: Causes of vitamin B12 and folate deficiency Causes of vitamin B12 deficiency Pernicious anaemia Nutritional (eg, vegan diet) Blind loops, bacterial overgrowth Ileal disease Gastrectomy Fish tapeworm (Diphyllobothrium latum) Causes of folate deficiency Dietary (eg, elderly people, alcoholic patients) Malabsorption (eg, coeliac disease, Crohn’s disease) Drugs (eg, anticonvulsants) Folate antagonists (eg, methotrexate, trimethoprim) Excessive loss/use (eg, pregnancy, haemolysis)
cases), although the MCV may be lower if there is coexisting iron deficiency, thalassaemia trait, inflammatory disease, or red-cell fragmentation.18 The blood film (figure 4) shows features of dyserythropoiesis, including oval macrocytosis, red-cell basophilic stippling, Howell-Jolly bodies, occasional circulating megaloblasts, and hypersegmented neutrophils. The white-cell and platelet counts may be low. Bone-marrow examination (figure 4), although seldom necessary now since assays for vitamin B12 are readily available, shows megaloblastic change with pronounced erythroid hyperplasia and predominance of early erythroid precursors, open atypical nuclear chromatin patterns, mitotic figures, and ‘giant’ metamyelocytes. Bone-marrow storage iron and serum ferritin are increased in most cases,19 the serum concentration of vitamin B12 is low, and serum and redcell folate concentrations are generally normal. Lactate dehydrogenase concentrations are high owing to ineffective erythropoiesis and early destruction of red cells.20 In western societies pernicious anaemia is the commonest cause of vitamin B12 deficiency (panel 2),21 with a rate of one in 10 000 in northern Europeans. Pernicious anaemia is due to autoimmune gastric atrophy and loss of intrinsic-factor production required for normal absorption of vitamin B12. Investigation and management Blood concentrations of both vitamins can be measured by ELISA methods.22 Vitamin B12 is measured as serum vitamin B12; folate may be measured as serum and redcell folate, the latter providing an estimate of body folate stores. Additional investigations in vitamin B12 deficiency include the measurement of serum intrinsic factor and parietal-cell antibodies, upper gastrointestinal endoscopy with duodenal biopsy, small-bowel barium studies, and other tests for malabsorption. Malabsorption of vitamin B12 can be tested by a urinary excretion method, such as the Schilling test,23 or whole-body B12 counting.24 Standard therapy consisting of five to six intramuscular injections of hydroxocobalamin (1 mg each) over a 3-week period corrects the haematological abnormalities in vitamin B12 deficiency and replaces stores. Patients are then given 1 mg intramuscularly every 3 months for life. Administration of folic acid in a patient with vitamin B12 deficiency may induce a haematological response but will worsen any neurological symptoms, and can actually precipitate subacute combined degeneration of the cord. In folate deficiency the serum and red-cell folate concentrations are low. Parietal-cell and intrinsic-factor autoantibodies are rarely present. Treatment consists of
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An acquired cause for haemolysis is likely if the patient presents for the first time in adult life, if previous blood counts have been normal, if there is no family history of haemolytic anaemia, or if the patient has recently been systemically unwell or has begun treatment with a new drug. There are many causes of haemolytic anaemia. Although specific diagnostic features may be present, there are features shared with many forms of haemolysis including: blood-film abnormalities such as polychromasia, spherocytosis, red-cell fragments, reticulocytosis, raised concentrations of serum bilirubin (unconjugated), urinary urobilinogen, and lactate dehydrogenase, and low or absent serum haptoglobins. Red-cell destruction can take place in the reticuloendothelial system (extravascular haemolysis) or in the circulation (intravascular haemolysis); the latter is associated with haemoglobinaemia, haemoglobinuria, haemosiderinuria, and methaemalbuminaemia, and follows, for example, ABO-mismatched transfusion. Extravascular haemolysis occurs in autoimmune haemolytic anaemias, rhesus-incompatible transfusion, and many chronic haemolytic disorders. The haemolysis can be immune or non-immune in origin. The direct antiglobulin test is positive in many cases with immune causes; positive reactions with specific reagents such as anti-IgG and anti-C3 suggest the presence of “warm” (IgG) or “cold” (IgM predominantly) antibodies, respectively. Investigation should also aim to find out whether an underlying disorder is causing the haemolysis or whether it is idiopathic. Underlying lymphoproliferative disorders such as chronic lymphocytic leukaemia, sepsis, renal or liver disease, and recent blood transfusion should be sought. Figure 5: Autoimmune haemolytic anaemia (warm, IgG antibody) RBC=red blood cell; reticulocyte=polychromatic (blue) RBC.
folic acid 5 mg daily for 4 months to replace stores. If there is an underlying cause such as coeliac disease, it should be treated. Folate prophylaxis Women planning pregnancy are advised to take 400 g folic acid daily before conception and until 12 weeks of pregnancy to prevent neural-tube defects (5 mg/day for women with a previous affected pregnancy).25 Folate fortification of cereal grains at 1·4 mg/kg has been made mandatory in the USA as an additional method of improving the folate status of the population. Prophylactic folate is also recommended in other states of increased demand such as long-term haemodialysis and chronic haemolytic disorders.26
Haemolytic anaemias due to reduced red-cell lifespan Acquired haemolytic disorders Haemolysis describes any situation in which the red-cell lifespan is less than 120 days. Anaemia results if there is failure of a compensatory marrow response. The predominant site of red-cell destruction is the red pulp of the spleen but other reticuloendothelial tissues are also involved.
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Autoimmune haemolytic anaemia Autoimmune haemolytic anaemia involves the production of red-cell autoantibodies, which may be warm or cold. Red cells react with autoantibody, with or without complement, causing their premature destruction by the reticuloendothelial system. The spleen and marrow ingest antibody-coated red cells, but in cases of incomplete phagocytosis spherical red cells are released into the peripheral circulation as spherocytes. Most warm autoimmune haemolytic anaemias are idiopathic, but this disorder may also be found in patients with underlying lymphoid malignant disorders or infections or in patients receiving certain classes of drugs.27,28 Presentation can be at any age but is commoner in adults. A full blood count shows a low concentration of haemoglobin, with spherocytes in the blood film (figure 5). The MCV is high in many cases, owing to the presence of reticulocytosis. Serological assays confirm the presence of IgG on the red-cell surface (by the direct antiglobulin test). Prednisolone 1 mg/kg daily for 1–2 weeks is the standard treatment; after this time the prednisolone dose is tailed off. The haemoglobin concentration rises and the reticulocyte count falls towards normal during this period. In cases of relapse other methods of immunosuppression may be used, including intravenous immunoglobulin. Splenectomy may be beneficial in some patients who do not respond to standard treatment. Cold autoimmune haemolytic anaemia occurs after infections (eg, Mycoplasma pneumoniae infection, glandular
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fever, or cytomegalovirus infection), and involves the production of polyclonal IgM autoantibodies. In elderly patients cold haemagglutinin disease is the result of a monoclonal IgM autoantibody. Investigations will confirm the presence of complement on the red-cell surface. Drug-induced haemolysis Drugs can induce haemolysis through various mechanisms,29 including: a hapten mechanism, in which the drug interacts with a red-cell component generating antigens that stimulate antibody production (eg, penicillins); autoantibody-mediated haemolysis, in which a warm autoantibody is generated (eg, -methyldopa); or an innocent-bystander mechanism in which the drug forms an immune complex with antibody that then attaches to the red cells leading to complement fixation and red-cell destruction (eg, quinine and quinidine). In addition, drugs can shorten the red-cell lifespan through interference with membrane lipids or through oxidation of haemoglobin. Non-immune red-cell destruction Physical agents can induce red-cell destruction through burns, damage on prosthetic heart valves, hypersplenism, haemolytic uraemic syndrome/thrombotic thrombocytopenic purpura, and march haemoglobinuria (in which red cells are destroyed in the soles of the feet of runners or soldiers).30
Anaemia due to clonal stem-cell disorders Myelodysplastic syndromes These syndromes are a morphologically and clinically heterogeneous group of acquired clonal bone-marrow stem-cell disorders predominantly affecting elderly people, although they may occur in younger adults and rarely in children.31 Myelodysplastic syndromes are characterised by ineffective bone-marrow haemopoiesis; dysplastic and defective blood cells are destroyed by the marrow before they ever enter the peripheral circulation, with resultant peripheral-blood cytopenias. The syndrome can be primary, arising de novo, or secondary to the administration of alkylating drugs including melphalan, chlorambucil, cyclophosphamide, and busulphan. Oncogene mutations have been reported in myelodysplastic syndromes; for example ras mutations have been detected in 9–40% cases and p53 mutations in 5–10%, although the role played by such mutations is unknown. The incidence of primary myelodysplastic syndromes increases with age—the annual incidence is 4·9 per 100 000 in the 50–70-year age-group and 22·8 per 100 000 in people older than 70. Although myelodysplastic syndromes were previously termed preleukaemic syndromes, many patients survive for many years with no evidence of development of leukaemia. Myelodysplastic syndromes are diagnosed by chance in about 20% of patients, when they have full blood counts done for other reasons. However, most patients present with symptoms reflecting the underlying peripheral-blood cytopenias—namely, anaemia, infection (caused by neutropenia or dysfunctional neutrophils) and bruising or bleeding (thrombocytopenia or dysfunctional platelets). The diagnosis is based on the blood count in conjunction with characteristic abnormalities of red and white blood cells and megakaryocytes in the bone marrow. The French-American-British classification32
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divides myelodysplastic syndromes into five subtypes based on bone-marrow morphology, immature (blast) cell count in blood and marrow, presence of iron-laden ring sideroblasts, and other cytological features. The subtypes are: refractory anaemia, so-called because these marrow disorders do not respond to iron or other haematinic replacement therapy; refractory anaemia with ring sideroblasts; refractory anaemia with excess blasts; refractory anaemia with excess blasts in transformation; and chronic myelomonocytic leukaemia. Refractory anaemia and refractory anaemia with ring sideroblasts have a good prognosis with median survival of 50 months. Refractory anaemia with excess blasts in transformation has a much poorer prognosis and median survival of only 6 months, largely due to the fact that this form, and refractory anaemia with excess blasts, are associated with a much higher leukaemic blast-cell count in the marrow and have a greater propensity to progress to frank acute myeloid leukaemia. Anaemia with a reduction in white-blood-cell and platelet counts is common. The blood film may show redcell abnormalities including variation in size and shape of red cells, macrocytosis, and basophilic stippling. Neutrophils have fewer cytoplasmic granules than normal (hypogranular) and have characteristic abnormalities of the nucleus. Despite the peripheral-blood cytopenias typical of myelodysplastic syndromes, the bone marrow is hypercellular in many cases, reflecting the ineffective haemopoiesis associated with the disorders. Acquired (ie, non-constitutional) chromosomal abnormalities are common in myelodysplastic syndromes, and analysis of bone-marrow chromosomes provides useful prognostic information. For example, patients with normal karyotypes or loss of the long arm of chromosome 5 (5q abnormality33) have a far better prognosis than those with loss of chromosome 7, deletions of 7q, or more complex karyotypes. Acquired chromosomal abnormalities are present in at least 50% of patients with primary myelodysplastic syndromes, and are more common in poor-prognosis (>60% patients with refractory anaemia with excess blasts in transformation) than in good-prognosis myelodysplastic syndromes (<30% in refractory anaemia). Chromosome abnormalities associated with poor prognosis include loss of chromosome 7 (-7), deletion of the long arm of chromosome 7 (del7q) or short arm of chromosome 17 (del17p), complex abnormalities involving multiple chromosomes, and evolving chromosome abnormalities particularly if the chromosomes were previously normal. Most patients with myelodysplastic syndromes are elderly and the management is essentially supportive. Red-cell transfusions are given when patients have symptoms caused by their anaemia. Thrombocytopenic bleeding can be helped by regular platelet transfusion. Infection is the main cause of morbidity and mortality in patients with myelodysplastic syndromes. Patients who develop infection should be treated promptly with antibiotics. Bone-marrow transplantation is an option for younger patients (eg, those younger than 50 years).34 Paroxysmal nocturnal haemoglobinuria Paroxysmal nocturnal haemoglobinuria is a rare acquired clonal disorder of the haemopoietic stem cell, characterised by chronic intravascular haemolysis, thrombotic tendency, and variable degrees of bone-
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marrow failure. The underlying defect is somatic mutation of the X-linked gene for phosphatidylinositol glycan protein A; this protein is required for production of glycerylphosphatidylinositol, which protects the red-cell membrane against complement-mediated damage. Other proteins implicated in paroxysmal nocturnal haemoglobinuria include decay-accelerating factor (or CD55) and CD59; when these proteins are absent, complement-mediated red-cell lysis occurs. The disorder affects young people but may be diagnosed in childhood or later life. The patient complains of passing dark urine, usually on waking. The haemoglobin concentration is low, and the MCV is high (caused by reticulocytosis, bone-marrow failure, or both). The white-cell and platelet counts are low in many cases. Other features of haemolysis are commonly present, including raised serum lactate dehydrogenase and bilirubin, with absence of haptoglobins. Chronic haemoglobinuria and haemosiderinuria lead to high urinary iron loss and ultimately iron deficiency. Together with these features, a positive Ham’s acidified serum test supports the diagnosis of paroxysmal nocturnal haemoglobinuria. Flow-cytometric analysis of red cells with anti-CD55 and anti-CD59 has replaced the Ham’s test. The increased risk of thrombosis, which may affect mesenteric, hepatic (Budd-Chiari syndrome), or other veins, is probably due to complement-mediated activation of platelets deficient in CD55 and CD59. The median survival is 10 years or longer, and the disorder has a close association with aplastic anaemia. Serious thrombosis occurs in 20% of patients, and a small proportion (5%) show progression to acute leukaemia. Treatment is essentially supportive and includes prompt treatment of infection, red-cell transfusion, and folic acid and iron supplementation. Thrombotic complications require vigorous treatment followed by life-long oral anticoagulation. In patients with severe bone-marrow failure, treatment options include immunosuppression with antilymphocyte globulin or cyclosporin, or siblingdonor bone-marrow transplantation. Bone-marrow transplantation from a matched unrelated donor may be an option for the treatment of severe paroxysmal nocturnal haemoglobinuria in patients younger than 25 years.35,36 Aplastic anaemia Aplastic anaemia, one form of bone-marrow-failure syndrome, describes the presence of peripheral-blood pancytopenia with a grossly hypocellular bone marrow, in which there is replacement of normal marrow haemopoietic cells by fat cells.37 By definition, there are no cancerous, leukaemic or other abnormal cells in either blood or marrow. There is evidence that aplastic anaemia is caused by an intrinsic defect of the haemopoietic stem cells that results in variable degrees of stem-cell loss. For example, syngeneic (identical twin) bone-marrow transplantation, without the use of immunosuppression, results in cure in 50% of cases.38 Other mechanisms invoked in the pathogenesis of aplastic anaemia include abnormalities of the bone-marrow stromal microenvironment, immune suppression of the marrow, and growth-factor deficiency. Since patients show variable responses to treatment, and the disease evolution is diverse, aplastic anaemia probably represents a heterogeneous group of disorders, rather than a single disease entity.
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Aplastic anaemia can be hereditary (for example, Fanconi anaemia, Diamond-Blackfan syndrome) or acquired; the latter includes the inevitable aplasia that follows cytotoxic chemotherapy or radiotherapy; idiosyncratic (drugs); postviral (hepatitis, cytomegalovirus, Epstein-Barr virus); and immune-mediated (for example, systemic lupus erythematosus). Around two-thirds of cases of aplastic anaemia are idiopathic, with no obvious underlying cause. Idiopathic aplastic anaemia is rare, with an annual incidence of two to five cases per million population (although the disorder is ten times more common in Oriental populations). Some patients have few symptoms at presentation and others have non-specific features including tiredness, bleeding, and a propensity to infection caused by decreased haemoglobin, platelets, and white blood cells, respectively. The full blood count shows variable degrees of pancytopenia with lower than normal haemoglobin concentration, white-cell count, and platelet count and a blood film that lacks major pathological features. Reticulocytes (young red blood cells) are low in number or absent from peripheral blood. The bone-marrow aspirate and trephine biopsy show replacement of normal haemopoietic tissue by fat cells, and the few haemopoietic cells that remain are lymphocytes and plasma cells. The Ham’s acidified serum lysis test is negative. Idiopathic aplastic anaemia can be stable, progressive, or unstable.37 Patients with severe forms (neutrophils <0·5109/L, platelets <20109/L, reticulocytes <1%) have a one in ten chance of surviving 1 year from diagnosis with only supportive treatment. Patients in whom the pancytopenia is less pronounced fare somewhat better. The progressive group has pancytopenia that worsens with time. Finally, the unstable group may develop abnormal clonal marrow cell populations. For example, by means of laboratory tests such as the Ham’s test, paroxysmal nocturnal haemoglobinuria clones are found in 10–20% of patients with prolonged idiopathic aplastic anaemia, although only a minority develop florid paroxysmal nocturnal haemoglobinuria. Other abnormal clones include acute myeloid leukaemia and myelodysplastic syndromes. Management consists of supportive therapy and treatment aimed at restoring normal bone-marrow activity. Supportive therapy involves red-cell and platelet transfusions, prophylactic antibiotics and antifungals, and general hygiene measures. If patients are treated solely in this way, only 28% are alive at 2 years from diagnosis.39 Specific measures to restore marrow activity include the use of immunosuppression, such as antilymphocyte globulin, androgens (eg, oxymethalone), and haemopoietic growth factors such as granulocyte colonystimulating factor. Sibling-donor bone-marrow or stemcell transplantation, offering long-term survival of more than 65%, should be considered in patients younger than 50 years who have suitable donors. Over a period of 10 years up to 40% of patients who respond to immunosuppression may develop paroxysmal nocturnal haemoglobinuria, myelodysplasia, or acute myeloid leukaemia.40
Acquired polycythaemia Polycythaemias The polycythaemias are red-cell disorders associated with an increase in haemoglobin concentration, packed-cell
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Panel 3: Erythropoietin concentrations in secondary polycythaemias Appropriate erythropoietin increase Hypoxia Chronic lung disease Heavy smoking (carboxyhaemoglobin) High altitude Congenital cyanotic heart disease Gross obesity High-affinity haemoglobin (familial erythrocytosis) Inappropriate erythropoietin increase Renal disorders Hypernephroma Hydronephrosis Polycystic kidney disease Post-renal transplant Other Hepatoma Uterine leiomyoma Cerebellar haemangioblastoma
volume, and red-cell count, and are subdivided into absolute erythrocytosis (true increase in red-cell mass) and relative erythrocytosis (red-cell mass normal but plasma volume reduced). A red-cell mass greater than 25% above the predicted value indicates absolute polycythaemia. Three main groups are recognised: primary proliferative polycythaemia (also called polycythaemia rubra vera),41 secondary polycythaemia (due to appropriate or inappropriate increases in erythropoietin concentration), and apparent polycythaemia42 (older terms include relative, spurious, and Gaisbock’s syndrome). Only the first two groups have a true increase in red-cell mass. Primary polycythaemia Polycythaemia rubra vera is one of the myeloproliferative disorders, a group of non-malignant clonal bone-marrow stem-cell disorders that includes essential thrombocythaemia, idiopathic myelofibrosis, and chronic myeloid leukaemia. The underlying cause is unknown but is likely to be acquired genetic changes in the stem cell leading to disturbance of the normal cellular growth pattern. Cellculture studies have shown that the red-cell precursors are able to grow independently of erythropoietin and are more sensitive than normal to various growth factors including erythropoietin and thrombopoietin. Cellular proliferation is believed to be driven through protein phosphorylation, and the tumour suppressor gene, SHP-1, seems to be a suitable candidate in this process; the SHP-1 product interacts with several growth-factor receptors including that of erythropoietin. In addition, loss of the SHP-1 binding domain from the erythropoietin receptor results in one form of congenital polycythaemia.43 Polycythaemia rubra vera is a disorder of older people (median age 55–60 years) of both sexes; it may be a chance finding, or patients may present with vascular or other complications including digital ischaemia, cerebrovascular accident, transient ischaemic attack, myocardial infarction, bleeding, bruising, hyperuricaemia/gout, or itching. The full blood count shows increased haemoglobin concentration, packed-cell volume, and red-cell count; 70% of cases have high white-blood-cell and platelet counts. The MCV may be low owing to concomitant iron deficiency caused through, for example, gastrointestinal bleeding. The red-cell mass is higher than normal (>36
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mL/kg in men; >32 mL/kg in women). The bone marrow is hypercellular with increased red cells and megakaryocytes, though this feature is not diagnostic in isolation. Chromosome studies are not carried out routinely but previous studies have shown chromosomal abnormalities in 15% of patients, including trisomy 1, 8, and 9, and deletion of 13q and 20q. The packed-cell volume is decreased by removal of 1 unit of blood every alternate day until the packed-cell volume is within desired range, then every 6–8 weeks. Alternatively, the packed-cell volume can be controlled by use of oral chemotherapy, such as hydroxyurea, an inhibitor of ribonucleotide reductase. Busulphan is no longer the drug of choice because it induces secondary leukaemias in later life and pulmonary fibrosis. Phosphorus-32, reserved for use in elderly patients, may provide control of packed-cell volume for up to 18 months. Polycythaemia rubra vera may progress to myelofibrosis (30%) or acute myeloid leukaemia (5%) with a median survival of 10–16 years. Secondary polycythaemias These disorders are predominantly driven by hypoxia, resulting in a physiologically appropriate compensatory increase in erythropoietin production; a smaller proportion are caused by inappropriate erythropoietin production (panel 3). The complications are similar to those of polycythaemia rubra vera but there is no progression to myelofibrosis or acute leukaemia. Treatment aims to reduce the packed-cell volume to a reasonably safe value, but care should be taken in patients whose increase in red-cell mass is compensatory, since over-vigorous treatment may be detrimental. Apparent polycythaemia This term describes patients with increased haemoglobin concentration and packed-cell volume but normal red-cell mass. A reduction in plasma volume causes the erythrocytosis. Apparent polycythaemia is more common than polycythaemia rubra vera and typically affects middle-aged men who are obese and hypertensive, who smoke, and who have high alcohol consumption. In some cases apparent polycythaemia may be associated with diuretic therapy. Treatment is that of the underlying disorder. The patient should reduce weight and cigarette consumption. Venesection to a packed-cell volume of less than 50% may be required in patients with definite cardiovascular risks. References 1 Cook JD, Skikne BS, Baynes RD. Iron deficiency: the global perspective. Adv Exp Med Biol 1994; 356:219–28. 2 Provan D. Mechanisms and management of iron deficiency anaemia. Br J Haematol 1999; 105 (suppl 1):19–26. 3 Gunshin H, Mackenzie B, Berger UV, et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997; 388:482–88. 4 Seligman PA, Schleicher RB, Allen RH. Isolation and characterization of the transferrin receptor from human placenta. J Biol Chem 1979; 254:9943–46. 5 Kohgo Y, Niitsu Y, Nishisato T, et al. Quantitation and characterization of serum transferrin receptor in patients with anemias and polycythemias. Jpn J Med 1988; 27:67–40. 6 Zoller H, Pietroangelo A Vogel W, Weiss G. Duodenal metal transporter (DMT-1, NRAMP-2) expression in patients with hereditary haeochromatosis. Lancet 1999; 353:2120–23. 7 Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996; 13:399–408.
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HAEMATOLOGY 8 Waheed A, Parkkila S, Saarnio J, et al. Association of HFE protein with transferrin receptor in crypt enterocytes of human duodenum. Proc Natl Acad Sci USA 1999; 96:1579–84. 9 Gray NK, Hentze MW. Iron regulatowry protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs. EMBO J 1994; 13:3882–91. 10 Kaptain S, Downey WE, Tang C, et al. A regulated RNA binding protein also possesses aconitase activity. Proc Natl Acad Sci USA 1991; 88:10109–13. 11 Mullner EW, Kuhn LC. A stem-loop in the 3’ untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm. Cell 1988; 53:815–25. 12 Means RT, Krantz SB. Progress in understanding the pathogenesis of the anemia in chronic disease. Blood 1992; 80:1639–47. 13 Baer AN, Dessypris EN, Krantz SB. The pathogenesis of anemia in rheumatoid arthritis: a clinical and laboratory analysis. Semin Arthritis Rheum 1990; 19:209–23. 14 Pippard MJ, Hoffbrand AV. Iron. In: Hoffbrand AV, Lewis SM, Tuddenham EGD, eds. Postgraduate haematology, 4th edn. Oxford: Butterworth-Heinemann, 1999: 23–46. 15 Wickramasinghe SN. Morphology, biology and biochemistry of cobalamin-and folate-deficient bone marrow cells. Baillieres Clin Haematol 1995; 8:441–59. 16 Savage DG, Lindenbaum J. Neurological complications of acquired cobalamin deficiency: clinical aspects. Baillieres Clin Haematol 1995; 8: 657–78. 17 Molloy AM, Daly S, Mills JL, et al. Thermolabile variant of 5,10methylenetetrahydrofolate reductase associated with low red-cell folates: implications for folate intake recommendations. Lancet 1997; 349:1591–93. 18 Spivak JL. Masked megaloblastic anemia. Arch Intern Med 1982; 142: 2111–14. 19 Hussein S, Laulicht M, Hoffbrand AV. Serum ferritin in megaloblastic anaemia. Scand J Haematol 1978; 20:241–45. 20 Winston RM, Warburton FG, Stott A. Enzymatic diagnosis of megaloblastic anaemia. Br J Haematol 1970; 19:587–92. 21 Toh BH, van Driel IR, Gleeson PA. Pernicious anemia. N Engl J Med 1997; 337:1441–48. 22 Chanarin I, Metz J. Diagnosis of cobalamin deficiency: the old and the new. Br J Haematol 1997; 97:695–700. 23 Fairbanks VF, Wahner HW, Phyliky RL. Tests for pernicious anemia: the Schilling test. Mayo Clin Proc 1983; 58:541–44. 24 Callender ST, Witts LJ, Warner GT, Oliver R. The use of a simple whole-body counter for haematological investigations. Br J Haematol 1966; 12:276–82. 25 Department of Health, Expert Advisory Group. Folic acid and the prevention of neural tube defect. London: Department of Health, 1992.
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26 Hoffbrand AV. Megaloblastic anaemia. In: Hoffbrand AV, Lewis SM, Tuddenham EGD, eds. Postgraduate haematology, 4th edn. Oxford: Butterworth-Heinemann, 1999: 47–67. 27 Dacie JV, ed. Autoimmune haemolytic anaemias. Edinburgh: Churchill Livingstone, 1992. 28 Petz LD. Drug-induced autoimmune hemolytic anemias. Transfus Med Rev 1993; 7:242–54. 29 Gordon-Smith EC, Hows JM, Luzzatto L. Acquired haemolytic anaemias. In: Hoffbrand AV, Lewis SM, Tuddenham EGD, eds. Postgraduate haematology, 4th edn. Oxford: Butterworth-Heinemann, 1999: 144–63. 30 Hallberg L, Magnusson B. The etiology of “sports anemia”: a physiological adaptation of the oxygen-dissociation curve of hemoglobin to an unphysiological exercise load. Acta Med Scand 1984; 216:147–8. 31 Passmore SJ, Hann IM, Stiller CA, et al. Pediatric myelodysplasia: a study of 68 children and a new prognostic scoring system. Blood 1995; 85:1742–50. 32 Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982; 51:189–99. 33 Boultwood J, Lewis S, Wainscoat JS. The 5q- syndrome. Blood 1994; 84:3253–60. 34 Anderson JE, Anasetti C, Appelbaum FR, et al. Unrelated donor marrow transplantation for myelodysplasia (MDS) and MDS-related acute myeloid leukaemia. Br J Haematol 1996; 93:59–67. 35 Rosse WF, Ware RE. The molecular basis of paroxysmal nocturnal hemoglobinuria. Blood 1995; 86:3277–86. 36 Socie G, Mary JY, de Gramont A, et al. Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. Lancet 1996; 348:573–77. 37 Gordon-Smith EC, Lewis SM. Aplastic anaemia and other types of bone marrow failure. In: Hoffbrand AV, Lewis SM, Tuddenham EGD, eds. Postgraduate haematology. 4th edn. Oxford: ButterworthHeinemann, 1999: 68–90. 38 Champlin RE, Feig SA, Sparkes RS, Galen RP. Bone marrow transplantation from identical twins in the treatment of aplastic anaemia: implication for the pathogenesis of the disease. Br J Haematol 1984; 56:455–63. 39 Camitta BM, Thomas ED, Nathan DG, et al. A prospective study of androgens and bone marrow transplantation for treatment of severe aplastic anemia. Blood 1979; 53:504–14. 40 de Planque MM, Kluin-Nelemans HC, van Krieken HJ, et al. Evolution of acquired severe aplastic anaemia to myelodysplasia and subsequent leukaemia in adults. Br J Haematol 1988; 70:55–62. 41 Berlin NI. Polycythemia vera. Semin Hematol 1997; 34:1–80. 42 Pearson TC. Apparent polycythaemia. Blood Rev 1991; 5:205–13. 43 Bench AJ, Huntly BJP, Nacheva EP, Champion KM, Green AR. Myeloproliferative disorders. In: Provan D, Gribben JG, eds. Molecular haematology. Oxford: Blackwell Science, 2000.
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