Hussien Mohammed Jumaah CABM Lecturer in internal medicine Mosul College of Medicine Monday , 11 April , 2016
anaemias
Around 30% of the total world population is anaemic and half of these, have iron deficiency. The classification of anaemia by the size of the red cells (MCV) indicates the likely cause . If red cells cannot acquire haemoglobin at a normal rate, they will undergo more divisions than normal and will have a low MCV when finally released into the blood. The MCV is low because component parts of the haemoglobin molecule are not fully available: that is, iron in iron deficiency, globin chains in thalassaemia, haem ring in congenital sideroblastic anaemia and, occasionally, poor iron utilisation in the anaemia of chronic disease.
Factors which influence the size of red cells in anaemia. In microcytosis, the MCV is < 76 fL. In macrocytosis, the MCV is > 100 fL. (MCV = mean cell volume; RBC = red blood cell)
Causes of red cells with a raised MCV.Megaloblastic anaemiaCytotoxic drugsMyelodysplasia . cells haemoglobinise normally but undergo fewer cell divisions, resulting in raised MCV. Others : The red cell membrane is composed of a lipid bilayer which will freely exchange with the plasma pool of lipid. Conditions such as liver disease, hypothyroidism, hyperlipidaemia and pregnancy are associated with raised lipids and may also cause a raised MCV. Reticulocytes are larger than mature red cells, so when the reticulocyte count is raised – for example, in haemolysis – this may also increase the MCV.
Iron deficiency anaemia This occurs when iron losses or physiological requirements exceed absorption. Blood loss The most common explanation is gastrointestinal blood loss. This may result from occult gastric or colorectal malignancy, gastritis, peptic ulceration, inflammatory bowel disease, diverticulitis, polyps and angiodysplastic lesions. Worldwide, hookworm and schistosomiasis are the most common causes of gut blood loss .
Iron deficiency anaemia Blood loss (continued) GI blood loss may be exacerbated by the chronic use of aspirin or NSAIDs, which cause intestinal erosions and impair platelet function. In women of child-bearing age, menstrual blood loss, pregnancy and breastfeeding contribute to iron deficiency by depleting iron stores. Very rarely, chronic haemoptysis or haematuria may cause iron deficiency.
Iron deficiency anaemia Malabsorption A dietary assessment should be made in all patients to ascertain their iron intake .Gastric acid is required to release iron from food and helps to keep iron in the soluble ferrous state . Achlorhydria in the elderly or that due to drugs such as proton pump inhibitors may contribute to the lack of iron availability from the diet, as may previous gastric surgery. Iron is absorbed actively in the upper small intestine and hence can be affected by coeliac.
Iron deficiency anaemia (continued) Physiological demands At times of rapid growth, such as infancy and puberty, iron requirements increase and may outstrip absorption. In pregnancy, iron is diverted to the fetus, the placenta and the increased maternal red cell mass, and is lost with bleeding at parturition .
Haematological physiology in pregnancy• Full blood count: increased plasma volume (40%) lowers normal Hb (reference range reduced to > 105 g/L at 28 wks). The MCV may increase. Gestational thrombocytopenia (rarely < 60 Ч 109/L) is a benign phenomenon.• Depletion of iron stores: iron deficiency is a common cause of anaemia, should be treated with oral iron supplement.• Vitamin B12: serum levels are physiologically low but deficiency is uncommon.• Folate: tissue stores may become depleted, and folate supplementation is recommended in all pregnancies.• Coagulation factors: from the second trimester, procoagulant factors increase approximately threefold, particularly fibrinogen, von Willebrand factor and factor VIII. This causes activated protein C resistance and a shortened activated partialthromboplastin time (APTT), and contributes to a prothrombotic state.• Anticoagulants: levels of protein C increase from the second trimester, while levels of free protein S fall as C4b binding protein increases.
The regulation of iron absorption, uptake and distribution in the body. The transport of iron is regulated in a similar fashion to enterocytes in other iron-transporting cells such as macrophages.
Iron deficiency anaemia InvestigationsConfirmation of iron deficiencyPlasma ferritin is a measure of iron stores in tissues and is the best single test to confirm iron deficiency. It is a very specific test; a subnormal level is due to iron deficiency or, very rarely, hypothyroidism or vitamin C deficiency. Ferritin levels can be raised in liver disease and in the acute phase response; in these conditions, a ferritin level of up to 100 μg/L may still be compatible with low bone marrow iron stores. Plasma iron and total iron binding capacity (TIBC)are measures of iron availability; hence they are affected by many factors besides iron stores.
Iron deficiency anaemia Investigations (continued) Investigation of the cause This will depend upon the age and sex of the patient, as well as the history and clinical findings. In men and in post-menopausal women with a normal diet, the upper and lower gastrointestinal tract should be investigated by endoscopy or radiological studies. Serum antiendomysial or anti- transglutaminase antibodies and possibly a duodenal biopsy are indicated to detect coeliac disease. In the tropics, stool and urine should be examined for parasites.
Iron deficiency anaemiaManagementUnless the patient has angina, heart failure or cerebral hypoxia, transfusion is not necessary and oral iron replacement is appropriate. Ferrous sulphate 200 mg 3 times daily (195 mg of elemental iron per day) is adequate and should be continued for 3–6 months to replete iron stores. Many patients suffer gastrointestinal side-effects with ferrous sulphate, including dyspepsia and altered bowel habit. When this occurs, reduction in dose to 200 mg twice daily or a switch to ferrous gluconate 300 mg twice daily (70 mg of elemental iron per day) should be tried.
Iron deficiency anaemiaManagement (continued) Delayed-release preparations are not useful, since they release iron beyond the upper small intestine, where it cannot be absorbed. The haemoglobin should rise by around 10 g/L every 7–10 days and a reticulocyte response will be evident within a week. A failure to respond adequately may be due to non-compliance, continued blood loss, malabsorption or an incorrect diagnosis. Patients with malabsorption or chronic gut disease may need parenteral iron therapy.
Iron deficiency anaemiaManagement (continued) Previously, iron dextran or iron sucrose wasused, but new preparations of iron isomaltose and iron carboxymaltose have fewer allergic effects and are preferred.Doses required can be calculated based on thepatient’s starting haemoglobin and body weight. Observation for anaphylaxis following an initial test dose is recommended.
Anaemia of chronic disease (ACD) Is a common type of anaemia, particularly in hospital populations. It occurs in the setting of chronic infection, chronic inflammation or neoplasia. The anaemia is not related to bleeding, haemolysis or marrow infiltration, is mild, with haemoglobin in the range of 85–115 g/L, and is usually associated with a normal MCV (normocytic, normochromic), though this may be reduced in long-standing inflammation.The serum iron is low but iron stores are normal orincreased, as indicated by the ferritin or stainablemarrow iron.
ACD : Pathogenesis The key regulatory protein that accounts for the findings characteristic of ACD is hepcidin, produced by the liver. Its production is induced by proinflammatory cytokines, especially IL-6. Hepcidin binds to ferroportin on the membrane of iron-exporting cells, such as small intestinal enterocytes and macrophages, internalising the ferroportin and thereby inhibiting the export of iron from these cells into the blood. The iron remains trapped inside the cells in the form of ferritin, levels of which are therefore normal or high in the face of significant anaemia. Inhibition or blockade of hepcidin is a potential target for treatment of this form of anaemia.
ACD Diagnosis and management It is often difficult to distinguish ACD associated with a low MCV from iron deficiency. Examination of the marrow may ultimately be required to assess iron stores directly. A trial of oral iron can be given in difficult situations. A positive response occurs in true iron deficiency but not in ACD. Measures which reduce the severity of the underlying disorder help to improve the ACD.
Investigations to differentiate anaemia of chronic disease from iron deficiency anaemia
Megaloblastic anaemia This results from a deficiency of vitamin B12 or folic acid, or from disturbances in folic acid metabolism. Folate is an important substrate of, and vitamin B12 a co-factor for, the generation of the essential amino acid methionine from homocysteine. This reaction produces tetrahydrofolate, which is converted to thymidine monophosphate for incorporation into DNA. Deficiency of either vitamin B12 or folate will therefore produce high plasma levels of homocysteine and impaired DNA synthesis. The end result is cells with arrested nuclear maturation but normal cytoplasmic development: nucleocytoplasmic asynchrony.Megaloblastic anaemia (continued) All proliferating cells will exhibit megaloblastosis; hence changes are evident in the buccal mucosa, tongue, small intestine, cervix, vagina and uterus. The high proliferation rate of bone marrow results in striking changes in the haematopoietic system. Cells arrested in development and die within the marrow; this ineffective erythropoiesis results in an expanded hypercellular marrow. The megaloblastic changes are most evident in the early nucleated red cell precursors, and haemolysis within the marrow results in a raised bilirubin and lactate dehydrogenase (LDH), but without the reticulocytosis characteristic of other forms of haemolysis. Iron stores are usually raised.
Megaloblastic anaemia (continued) The mature red cells are large and oval, and sometimes contain nuclear remnants. Nuclear changes are seen in the immature granulocyte precursors and a characteristic appearance is that of ‘giant’ metamyelocytes with a large ‘sausage-shaped’ nucleus. The mature neutrophils show hypersegmentation of their nuclei, with cells having six or more nuclear lobes. If severe, a pancytopenia may be present in the peripheral blood.
Megaloblastic anaemia (continued) Vitamin B12 deficiency, but not folate deficiency, is associated with neurological disease in up to 40% of cases. The main pathological finding is focal demyelination affecting the spinal cord, peripheral nerves, optic nerves and cerebrum. The most common manifestations are sensory, with peripheral paraesthesiae and ataxia of gait.
Vitamin B12 absorptionThe average daily diet contains 5–30 μg of vitamin B12,mainly in meat, fish, eggs and milk – well in excess ofthe 1 μg daily requirement. In the stomach, gastricenzymes release vitamin B12 from food and at gastric pH it binds to a carrier protein termed R protein. The gastric parietal cells produce intrinsic factor, a vitamin B12- binding protein which binds vitamin B12 at pH 8. As gastric emptying occurs, pancreatic secretion raises the pH and vitamin B12 released from the diet switches from the R protein to intrinsic factor.
Vitamin B12 absorption (continued) Bile also contains vitamin B12 which is available for reabsorption in the intestine. The vitamin B12–intrinsic factor complex binds to specific receptors in the terminal ileum, and vitamin B12 is actively transported by the enterocytes to plasma, where it binds to transcobalamin II, a transport protein produced by the liver, which carries it to the tissues for utilisation. The liver stores enough vitamin B12 for 3 years and this, together with the enterohepatic circulation, means that vitamin B12 deficiency takes years to become manifest, even if all dietary intake is stopped orsevere B12 malabsorption supervenes.
Vitamin B12 absorption (continued) Blood levels of vitamin B12 provide a reasonable indication of tissue stores and are usually diagnostic of deficiency. Levels of cobalamins fall in normal pregnancy. Spuriously low B12 values occur in women using the oral contraceptive pill and in patients with myeloma, in whom paraproteins can interfere with vitamin B12 assays.
Causes of vitamin B12 deficiencyDietary deficiencyThis only occurs in strict vegans.Gastric pathologyRelease of vitamin B12 from the food requires normalgastric acid and enzyme secretion, and this is impairedby hypochlorhydria in elderly patients or followinggastric surgery. Total gastrectomy invariably results inB12 deficiency within 5 years, often with iron deficiency; these patients need life-long 3-monthly vitamin B12 injections. After partial gastrectomy, B12 deficiency only develops in 10–20% by 5 years; an annual injection of vitamin B12 should prevent deficiency in this group.
Causes of vitamin B12 deficiency (continued) Pernicious anaemiaThis is an organ-specific autoimmune disorder in whichthe gastric mucosa is atrophic, with loss of parietal cellscausing intrinsic factor deficiency. In the absence ofintrinsic factor, less than 1% of dietary vitamin B12 isabsorbed. Pernicious anaemia has an incidence of25/100 000 population over the age of 40 years in developed countries, but an average age of onset of 60 years. It is more common in individuals with other autoimmune disease , hashimoto’s thyroiditis, Graves’disease, vitiligo, hypoparathyroidism or Addison’s or a family history of these or pernicious anaemia.
Pernicious anaemia (continued) The finding of anti-intrinsic factor antibodies in the context of B12 deficiency is diagnostic of pernicious anaemia without further investigation. Antiparietal cell antibodies are present in over 90% of cases but are also present in 20% of normal females over the age of 60 years; a negative result makes pernicious anaemia less likely but a positive result is not diagnostic. The Schilling test, involving measurement of absorption of radio-labelled B12 after oral administration before and after replacement of intrinsic factor, has fallen out of favour with the availability of autoantibody tests, greater caution in the use of radioactive tracers, and limited availability of intrinsic factor.
Causes of vitamin B12 deficiency (continued) Small bowel pathologyOne-third of patients with pancreatic exocrine insufficiency fail to transfer dietary vitamin B12 from R protein to intrinsic factor. This results in slightly low B12 values but no tissue evidence of vitamin B12 deficiency. Motility disorders or hypogammaglobulinaemia can result in bacterial overgrowth, and the ensuing competition for free vitamin B12 can lead to deficiency.This is corrected to some extent by appropriate antibiotics. A small number of people heavily infected with the fish tapeworm develop vitamin B12 deficiency.Inflammatory disease of the terminal ileum, such as Crohn’s disease, may impair the absorption of vitamin B12–intrinsic factor complex, as may surgery on that part of the bowel.
Folate absorptionFolates are produced by plants and bacteria; hencedietary leafy vegetables (spinach, broccoli, lettuce),fruits (bananas, melons) and animal protein (liver, kidney) are a rich source. An average Western diet contains more than the minimum daily intake of 50 μg but excess cooking destroys folates. Most dietary folate is present as polyglutamates; these are converted to monoglutamate in the upper small bowel and actively transported into plasma. Plasma folate is loosely bound to plasma proteins such as albumin and there is an Enterohepatic circulation. Total body stores of folate are small and deficiency can occur in a matter of weeks.
Folate deficiency The edentulous elderly or psychiatric patient is particularly susceptible to dietary deficiency and this is exacerbated in the presence of gut disease or malignancy. Pregnancy-induced folate deficiency is the most common cause of megaloblastosis worldwide and is more likely in the context of twin pregnancies, multiparity and hyperemesis gravidarum. Serum folate is very sensitive to dietary intake; a single folate-rich meal can normalise it in a patient with true folate deficiency, whereas anorexia, alcohol and anticonvulsant therapy can reduce it in the absence of megaloblastosis. For this reason, red cell folate levels are a more accurate indicator of folate stores and tissue folate deficiency.
Causes of folate deficiency
Clinical features of megaloblastic anaemiaNeurological findings in B12 deficiency
Investigations in megaloblastic anaemiaInvestigation of folic acid deficiency
Management of megaloblastic anaemia If a patient with a severe megaloblastic anaemia is very ill and treatment must be started before vitamin B12 and red cell folate results are available, that treatment should always include both folic acid and vitamin B12. The use of folic acid alone in the presence of vitamin B12 deficiency may result in worsening of neurological deficits. Rarely, if severe angina or heart failure is present, transfusion can be used in megaloblastic anaemia. The cardiovascular system is adapted to the chronic anaemia present in megaloblastosis, and the volume load imposed by transfusion may result in decompensation and severe cardiac failure. In such circumstances, exchange transfusion or slow administration of 1 U of red cells with diuretic cover may be given cautiously.Management of megaloblastic anaemia (continued) Vitamin B12 deficiencyTreated with hydroxycobalamin 1000 μg IM for 6 doses 2 or 3 days apart, followed by maintenance therapy of 1000 μg every 3 months for life.The reticulocyte count will peak by the 5th–10th day after starting replacement therapy. The haemoglobin will rise by 10 g/L every week until normalised. The response of the marrow is associated with a fall in plasma potassium levels and rapid depletion of ironstores. If an initial response is not maintained and the blood film is dimorphic (i.e. shows a mixture of microcytic and macrocytic cells), the patient may need additional iron therapy. A sensory neuropathy may take 6–12 months to correct; long-standing neurological damage may not improve.
Management of megaloblastic anaemia (continued) Folate deficiencyOral folic acid 5 mg daily for 3 weeks will treat acute deficiency and 5 mg once weekly is adequate maintenance therapy. Prophylactic folic acid in pregnancy prevents megaloblastosis in women at risk, and reduces the risk of fetal neural tube defects . Prophylactic supplementation is also given in chronic haematological disease associated with reduced red cell lifespan (e.g. haemolytic anaemias). There is some evidence that supraphysiological supplementation (400 μg/day) can reduce the risk of coronary and cerebrovascular disease by lowering plasma homocysteine levels. This has led the US Food and Drug Administration to introduce fortification of bread, flour and rice with folic acid.
Haemolytic anaemia Haemolysis indicates that there is shortening of the normal red cell lifespan of 120 days. To compensate, the bone marrow may increase its output of red cells six- to eightfold by increasing the proportion of red cells produced, expanding the volume of active marrow, and releasing reticulocytes prematurely. Anaemia only occurs if the rate of destruction exceeds this increased production rate. Red cell destruction overloads pathways for haemoglobin breakdown in the , causing a modest rise in unconjugated bilirubin in the blood and mild jaundice.
Haemolytic anaemia (continued) Increased reabsorption of urobilinogen from the gut results in an increase in urinary urobilinogen. Red cell destruction releases LDH into the serum. The bone marrow compensation results in a reticulocytosis, and sometimes nucleated red cell precursors appear in the blood. Increased proliferation of the bone marrow can result in a thrombocytosis, neutrophilia and, if marked, immature granulocytes in the blood, producing a leucoerythroblastic blood film.
Haemolytic anaemia (continued) The appearances of the red cells may give an indication of the likely cause of the haemolysis:• Spherocytes are small, dark red cells which suggest autoimmune haemolysis or hereditary spherocytosis.• Sickle cells suggest sickle-cell disease.• Red cell fragments indicate microangiopathic haemolysis.The compensatory erythroid hyperplasia may give rise to folate deficiency, with megaloblastic blood features.The differential diagnosis of haemolysis is determined by the clinical scenario in combination with the results of blood film examination and Coombs testing for antibodies directed against red cells.
Haemolytic anaemia (continued) Extravascular haemolysisPhysiological red cell destruction occurs in the reticuloendothelial cells in the liver or spleen, so avoiding free haemoglobin in the plasma. In most haemolytic states, haemolysis is predominantly extravascular.To confirm the haemolysis, patients’ red cells can belabelled with 51chromium. When re-injected, they can beused to determine red cell survival; when combinedwith body surface radioactivity counting, this indicate whether the liver or the spleen is the main source of red cell destruction. However, it is seldom performed in clinical practice.
Haemolytic anaemia (continued) Intravascular haemolysis Less commonly, red cell lysis occurs within the blood stream due to membrane damage by complement (ABO transfusion reactions, paroxysmal nocturnal haemoglobinuria), infections (malaria, Clostridium perfringens), mechanical trauma (heart valves, DIC) or oxidative damage (e.g. drugs such as dapsone and maloprim). When intravascular red cell destruction occurs, free haemoglobin is released into the plasma. Free haemoglobin is toxic to cells and binding proteins have evolved to minimise this risk.
Haemolytic anaemia Intravascular haemolysis (continued) Haptoglobin is an α2-globulin produced by the liver, which binds free haemoglobin, resulting in a fall in its levels during active haemolysis.Once haptoglobins are saturated, free haemoglobin isoxidised to form methaemoglobin, which binds toalbumin, in turn forming methaemalbumin, which canbe detected spectrophotometrically in the Schumm’stest. Methaemoglobin is degraded and any free haem isbound to a second binding protein called haemopexin.
Haemolytic anaemia Intravascular haemolysis (continued) If all the protective mechanisms are saturated, free haemoglobin may appear in the urine (haemoglobinuria). When fulminant, this gives rise to black urine, as in severe falciparum malaria infection . In smaller amounts, renal tubular cells absorb the haemoglobin, degrade it and store the iron as haemosiderin. When the tubular cells are subsequently sloughed into the urine, they give rise to haemosiderinuria, which is always indicative of intravascular haemolysis.
Causes of haemolysis. A Inherited causes. B Acquired causes. (CLL = chronic lymphatic leukaemia; DIC = disseminated intravascular coagulation; EBV = Epstein–Barr virus; G6PD = glucose-6-phosphate dehydrogenase; HUS = haemolytic uraemic syndrome; PK = pyruvate kinase; RA = rheumatoid arthritis; SLE = systemic lupus erythematosus; TTP = thrombotic thrombocytopenic purpura)
Investigation results indicating active haemolysis
Haemolytic anaemia (continued) Red cell membrane defectsThe basic structure is a cytoskeleton ‘stapled’ on to the lipid bilayer by special protein complexes.This structure ensures great deformability and elasticity; the red cell diameter is 8 μm but the narrowest capillaries in the circulation are in the spleen, measuring just 2 μm in diameter. When the normal red cell structure is disturbed, usually by a quantitative or functional deficiency of one or more proteins in the cytoskeleton, cells lose their elasticity. Each time such cells pass through the spleen, they lose membrane relative to their cell volume. This results in an increase in mean cell haemoglobin concentration (MCHC), abnormal cell shape and reduced red cell survival due to extravascular haemolysis.Haemolytic anaemia, Red cell membrane defects Hereditary spherocytosis This is usually inherited as an autosomal dominant condition, although 25% of cases have no family history and represent new mutations. The most common abnormalities are deficiencies of beta spectrin or ankyrin. The severity of spontaneous haemolysis varies. Most cases are associated with an asymptomatic compensated chronic haemolytic state with spherocytes present on the blood film, a reticulocytosis and mild hyperbilirubinaemia. Pigment gallstones are present in up to 50% of patients and may cause symptomatic cholecystitis. Occasional cases are associated with more severe haemolysis.
Haemolytic anaemia, Red cell membrane defectsHereditary spherocytosis (continued) The clinical course may be complicated by crises:• A haemolytic crisis occurs when the severity of haemolysis increases; this is rare, and usually associated with infection.• A megaloblastic crisis follows the development of folate deficiency; this may occur as a first presentation of the disease in pregnancy.• An aplastic crisis occurs in association with parvovirus B19 infection ,causes exanthem in children, temporarily switches off red cell production. Patients present with severe anaemia and a low reticulocyte count.
Haemolytic anaemia, Red cell membrane defects Hereditary spherocytosis (continued) Investigations The patient and other family members should be screened for features of compensated haemolysis. Haemoglobin levels are variable, depending on the degree of compensation. The blood film will show spherocytes but the direct Coombs test is negative, excluding immune haemolysis. An osmotic fragility test may show increased sensitivity to lysis in hypotonic saline solutions but is limited by lack of sensitivity and specificity. More specific flow cytometric tests, detecting binding of eosin-5-maleimide to red cells, are recommended in borderline cases.
Haemolytic anaemia, Red cell membrane defects Hereditary spherocytosis (continued) Management Folic acid prophylaxis, 5 mg daily, should be given for life. Consideration may be given to splenectomy, which improves but does not normalise red cell survival. Potential indications include moderate to severe haemolysis with complications (anaemia and gallstones), although splenectomy should be delayed until after 6 years of age in view of the risk of sepsis. Acute, severe haemolytic crises require transfusion support, blood must be cross-matched carefully and transfused slowly as haemolytic transfusion reactions may occur .
Haemolytic anaemia, Red cell membrane defects (continued) Hereditary elliptocytosis Group of disorders that produce an increase in elliptocytic red cells on the blood film and a variable degree of haemolysis. This is due to a functional abnormality of one or more anchor proteins in the red cell membrane, e.g. alpha spectrin or protein 4.1. Inheritance may be autosomal dominant or recessive. Hereditary elliptocytosis is less common than hereditary spherocytosis. The clinical course is variable and depends upon the degree of membrane dysfunction . Most cases present as an asymptomatic blood film abnormality, but occasional cases result in neonatal haemolysis or a chronic compensated haemolytic state. Management is the same as for hereditary spherocytosis.
Haemolytic anaemia Red cell enzymopathiesThe mature red cell must produce energy via ATP to maintain a normal internal environment and cell volume whilst protecting itself from the oxidative stress presented by oxygen carriage. Anaerobic glycolysis via the Embden–Meyerhof pathway generates ATP, and the hexose monophosphate shunt produces nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione to protect against oxidative stress. The impact of functional or quantitative defects in the enzymes in these pathways depends upon the importance of the steps affected and the presence of alternative pathways. In general, defects in the hexose monophosphate shunt pathway result in periodic haemolysis precipitated by episodic oxidative stress, whilst those in the Embden–Meyerhof pathway result in shortened red cell survival and chronic haemolysis.
Haemolytic anaemia Red cell enzymopathies (continued) Glucose-6-phosphate dehydrogenase deficiency (G6PD) The enzyme glucose-6-phosphate dehydrogenase is pivotal in the hexose monophosphate shunt pathway. Deficiencies result in the most common human enzymopathy, affecting 10% of the world’s population. The enzyme is a heteromeric structure made of catalytic subunits which are encoded by a gene on the X chromosome. The deficiency therefore affects males and rare homozygousfemales , but it is carried by females. Over 400 subtypes of G6PD are described. The most common types associated with normal activity are the B+ enzyme present in most Caucasians and 70% of Afro- Caribbeans , and the A+ variant present in 20% of Afro-Caribbeans. The two common variants associated with reduced activity are the A− variety in approximately 10% of Afro-Caribbeans, and the Mediterranean or B− variety
Haemolytic anaemia Red cell enzymopathies (G6PD) (continued) The deficiency in Caucasian and Oriental populations is more severe, with enzyme levels as low as 1%. Management aims to stop any precipitant drugs and treat any underlying infection. Acute transfusion support may be life-saving.
Glucose-6-phosphate dehydrogenase deficiency
Glucose-6-phosphate dehydrogenase deficiencyHaemolytic anaemia Red cell enzymopathies(continued) Pyruvate kinase deficiencyThis is the second most common red cell enzyme defect.It results in deficiency of ATP production and a chronichaemolytic anaemia. It is inherited as an autosomalrecessive trait. The extent of anaemia is variable; theblood film shows characteristic ‘prickle cells’ whichresemble holly leaves. Enzyme activity is only 5–20% ofnormal. Transfusion support may be necessary.
Haemolytic anaemia Red cell enzymopathies(continued) Pyrimidine 5′ nucleotidase deficiencyThe pyrimidine 5′ nucleotidase enzyme catalyses thedephosphorylation of nucleoside monophosphates and is important during the degradation of RNA in reticulocytes. It is inherited as an autosomal recessive trait and is as common as pyruvate kinase deficiency in Mediterranean, African and Jewish populations. The accumulation of excess ribonucleoprotein results in coarse basophilic stippling , associated with a chronic haemolytic state. The enzyme is very sensitive to inhibition by lead and this is the reason why basophilic stippling is a feature of lead poisoning.
Autoimmune haemolytic anaemia This results from increased red cell destruction due to red cell autoantibodies. The antibodies may be IgG or M, or more rarely IgE or A. If an antibody avidly fixes complement, it will cause intravascular haemolysis, but if complement activation is weak, the haemolysis will be extravascular. Antibody-coated red cells lose membrane to macrophages in the spleen and hence spherocytes are present in the blood.
Autoimmune haemolytic anaemia(continued) The optimum temperature at which the antibody is active (thermal specificity) is used to classify immune haemolysis:• Warm antibodies bind best at 37°C and account for80% of cases. The majority are IgG and often reactagainst Rhesus antigens.• Cold antibodies bind best at 4°C but can bind up to37°C in some cases. They are usually IgM and bindcomplement. To be clinically relevant, they must actwithin the range of normal body temperatures.They account for the other 20% of cases.
Autoimmune haemolytic anaemia(continued) Warm autoimmune haemolysis It occurs at all ages but is more common in middle age and in females. No underlying cause is identified in up to 50% of cases. The remainder are secondary to a wide variety of other . Investigations There is evidence of haemolysis and spherocytes on the blood film. The diagnosis is confirmed by the direct Coombs or antiglobulin test .
Autoimmune haemolytic anaemiaWarm autoimmune haemolysis (continued) The patient’s red cells are mixed with Coombs reagent, which contains antibodies against human IgG/M/complement. If the red cells have been coated by antibody in vivo, the Coombs reagent will induce their agglutination and this can be detected visually. The relevant antibody can be eluted from the red cell surface and tested against a panel of typed red cells to determine against which red cell antigen it is directed. The most common specificity is Rhesus and most often anti-e; this is helpful when choosing blood to cross-match.
Autoimmune haemolytic anaemia Warm autoimmune haemolysis (continued) The direct Coombs test can be negative in the presence of brisk haemolysis. A positive test requires about 200 antibody molecules to attach to each red cell; with a very avid complement-fixing antibody, haemolysis may occur at lower levels of antibodybinding. The standard Coombs reagent will miss IgA or IgE antibodies. Around 10% of all warm autoimmune haemolytic anaemias are Coombs test-negative.
Direct and indirect antiglobulin tests
Autoimmune haemolytic anaemiaWarm autoimmune haemolysis (continued) ManagementIf the haemolysis is secondary to an underlying cause, must be treated and any implicated drugs stopped. Prednisolone 1 mg/kg orally. A response is seen in 70–80% of cases but may take up to 3 weeks; a rise in haemoglobin will be matched by a fall in bilirubin, LDH and reticulocyte levels. Once the haemoglobin has normalised and the reticulocytosis resolved, the corticosteroid dose can be reduced slowly over about 10 weeks. Corticosteroids work by decreasing macrophage destruction of antibody coated red cells and reducing antibody production.Autoimmune haemolytic anaemia Warm autoimmune haemolysis Management (continued) Transfusion support may be required for life threatening problems, such as the development of heart failure or rapid unabated falls in haemoglobin. The least incompatible blood should be used but this may still give rise to transfusion reactions or the development of alloantibodies.
Autoimmune haemolytic anaemiaWarm autoimmune haemolysisManagement (continued) If the haemolysis fails to respond to corticosteroids or can only be stabilised by large doses, then splenectomy should be considered. This removes a main site of red cell destruction and antibody production, with a good response in 50–60% of cases. The operation can be performed laparoscopically with reduced morbidity. If splenectomy is not appropriate, alternative immunosuppressivetherapy with azathioprine or cyclophosphamide may be considered. This is least suitable for young patients, in whom long-term immunosuppression carries a risk of secondary neoplasms. The anti-CD20 (B cell) monoclonal antibody, rituximab, has shown some success in difficult cases.
Autoimmune haemolytic anaemia (continued) Cold agglutinin disease This is due to antibodies, usually IgM, which bind to the red cells at low temperatures and cause them to agglutinate. It may cause intravascular haemolysis if complement fixation occurs. This can be chronic when the antibody is monoclonal, or acute or transient when the antibody is polyclonal.
Autoimmune haemolytic anaemia Chronic cold agglutinin disease Affects elderly patients and may be associated with an underlying low-grade B cell lymphoma. It causes a low-grade intravascular haemolysis with cold, painful and often blue fingers, toes, ears or nose (acrocyanosis).The latter is due to red cell agglutination in the small vessels in these colder exposed areas. The blood film shows red cell agglutination and the MCV may be spuriously high because the automated analysers detect aggregates as single cells. Monoclonal IgM usually has anti-I or, less often, anti-i specificity. Treatment is directed at any underlying lymphoma but if idiopathic, then patients must keep extremities warm, especially in winter.
Autoimmune haemolytic anaemiaCold agglutinin disease (continued) Other causes of cold agglutinationCold agglutination can occur in association with Mycoplasmapneumoniae or with infectious mononucleosis.Paroxysmal cold haemoglobinuria is a very rare causeseen in children, in association with viral or bacterialinfection. An IgG antibody binds to red cells in theperipheral circulation but lysis occurs in the centralcirculation when complement fixation takes place. Thisantibody is termed the Donath–Landsteiner antibodyand has specificity against the P antigen on the red cells.
Alloimmune haemolytic anaemia Caused by antibodies against non-self red cells, and occurs after unmatched transfusion , or after maternal sensitisation to paternal antigens on fetal cells (haemolytic disease of the newborn).
Non-immune haemolytic anaemiaPhysical traumaPhysical disruption of red cells may occur in a numberof conditions and is characterised by the presence of redcell fragments on the blood film and markers of intravascular haemolysis:• Mechanical heart valves. High flow through incompetent valves or periprosthetic leaks through the suture ring holding a valve in place result in shear stress damage.• March haemoglobinuria. Vigorous exercise, such asprolonged marching or marathon running, cancause red cell damage in the capillaries in the feet.
Non-immune haemolytic anaemiaPhysical trauma (continued) • Thermal injury. Severe burns cause thermal damageto red cells, characterised by fragmentation and thepresence of microspherocytes in the blood.• Microangiopathic haemolytic anaemia. Fibrin deposition in capillaries can cause severe red celldisruption. It may occur in a wide variety ofconditions: disseminated carcinomatosis, malignantor pregnancy-induced hypertension, haemolyticuraemic syndrome , thrombotic thrombocytopenic purpura and disseminated intravascular coagulation .
Non-immune haemolytic anaemia (continued) Infection Plasmodium falciparum malaria may be associated with intravascular haemolysis; when severe, this is termed blackwater fever because of the associated haemoglobinuria. Clostridium perfringens septicaemia ,usually in the context of ascending cholangitis, may cause severe intravascular haemolysis with marked spherocytosis due to bacterial production of a lecithinase which destroys the red cell membrane. Chemicals or drugs Dapsone and sulfasalazine cause haemolysis by oxidative denaturation of haemoglobin. Denatured haemoglobin forms Heinz bodies in the red cells, visible on supravital staining with brilliant cresyl blue. Arsenic gas, copper, chlorates, nitrites and nitrobenzene derivatives may all cause haemolysis.
Paroxysmal nocturnal haemoglobinuria (PNH) a rareacquired, non-malignant clonal expansion of haematopoietic stem cells deficient in GPI-anchor protein; it results in intravascular haemolysis and anaemia because of increased sensitivity of red cells to lysis by complement.Episodes of intravascular haemolysis result in haemoglobinuria, most noticeable in early morning urine, which has a characteristic red–brown colour. The disease is associated with an increased risk of venous thrombosis in unusual sites, such as the liver or abdomen.
PNH (continued) PNH is also associated with hypoplastic bone marrow failure, aplastic anaemia and myelodysplastic syndrome. Management is supportive with transfusion and treatment of thrombosis. Recently, the anti-complement C5 monoclonal antibody eculizumab was shown to be effective in reducing haemolysis.
HaemoglobinopathiesThese diseases are caused by mutations affecting thegenes encoding the globin chains of the haemoglobinmolecule.Normal haemoglobin is comprised of two alpha and two non-alpha globin chains. α globin chains are produced throughout life, including in the fetus, so severe mutations may cause intrauterine death. Production of non-alpha chains varies with age; fetal haemoglobin (HbF-αα/ γγ) has two γ chains, while the predominant adult haemoglobin (HbA-αα/ββ) has two β chains. Thus, disorders affecting the beta chains do not present until after 6 months of age.
Haemoglobinopathies(continued) A constant small amount of haemoglobin A2 (HbA2-αα/δδ, usually < 2%) is made from birth.The haemoglobinopathies can be classified into qualitative or quantitative abnormalities.
Haemoglobinopathies(continued) Qualitative abnormalities – abnormal haemoglobinsIn qualitative abnormalities (called the abnormal haemoglobins), there is a functionally important alterationin the amino acid structure of the polypeptide chains ofthe globin chains. Several hundred such variants areknown; they were originally designated by letters of thealphabet, e.g. S, C, D or E, but are now described bynames usually taken from the town or district in whichthey were first described.
HaemoglobinopathiesQualitative abnormalities – abnormal haemoglobins (continued) The best-known example is haemoglobin S, found in sickle-cell anaemia. Mutations around the haem-binding pocket cause the haem ring to fall out of the structure and produce an unstable haemoglobin. These substitutions often change the charge of the globin chains, producing different electrophoretic mobility, and this forms the basis for the diagnostic use of haemoglobin electrophoresis to identify haemoglobinopathies.
Haemoglobinopathies (continued) Quantitative abnormalities – thalassaemiasIn quantitative abnormalities (the thalassaemias), thereare mutations causing a reduced rate of production ofone or other of the globin chains, altering the ratio ofalpha to non-alpha chains. In alpha- thalassaemia excess beta chains are present, whilst in beta- thalassaemia excess alpha chains are present. The excess chains precipitate, causing red cell membrane damage and reduced red cell survival.
Sickle-cell anaemia Sickle-cell disease results from a single glutamic acid to valine substitution at position 6 of the beta chain.It is inherited as an autosomal recessive trait . Homozygotes only produce abnormal beta chains that make haemoglobin S (HbS, termed SS), and this results in the clinical syndrome of sickle-cell disease. Heterozygotes produce a mixture of normal and abnormal beta chains that make normal HbA and HbS (termed AS), and this results in the clinically asymptomatic sickle-cell trait.
Sickle-cell anaemia (continued) Epidemiology The heterozygote frequency is over 20% in tropical Africa . Individuals with sickle-cell trait are relatively resistant to the lethal effects of falciparum malaria in early childhood. However, homozygous patients with sickle-cell anaemia do not have correspondingly greater resistance to falciparum malaria.
Sickle-cell anaemia (continued) PathogenesisWhen haemoglobin S is deoxygenated, the molecules of haemoglobin polymerise to form pseudocrystalline structures known as ‘tactoids’. These distort the red cell membrane and produce characteristic sickle-shaped cells . The polymerisation is reversible when re-oxygenation occurs. The distortion of the red cell membrane, however, may become permanent and the red cell ‘irreversibly sickled’.
Sickle-cell anaemia Pathogenesis (continued) The greater the concentration of sickle-cell haemoglobin in the individual cell, the more easily tactoids are formed, but this process may be enhanced or retarded by the presence of other haemoglobins.Thus, the abnormal haemoglobin C variant participates in the polymerisation more readily than haemoglobin A, whereas haemoglobin F strongly inhibits polymerisation.
Sickle-cell anaemia Clinical featuresSickling is precipitated by hypoxia, acidosis, dehydrationand infection. Irreversibly sickled cells have a shortened survival and plug vessels in the microcirculation. This results in a number of acute syndromes, termed ‘crises’, and chronic organ damage :• Painful vaso-occlusive crisis. Plugging of small vessels in the bone produces acute severe bone pain. This affects areas of active marrow: the hands and feet in children (so-called dactylitis) or the femora, humeri, ribs, pelvis and vertebrae in adults. Patients usually have a systemic response with tachycardia, sweating and a fever. This is the most common crisis.
Sickle-cell anaemia Clinical features (continued) • Sickle chest syndrome. This may follow a vasoocclusive crisis and is the most common cause of death in adult sickle disease. Bone marrow infarction results in fat emboli to the lungs, which cause further sickling and infarction, leading to ventilatory failure if not treated.
Sickle-cell anaemia Clinical features (continued) • Sequestration crisis. Thrombosis of the venous outflow from an organ causes loss of function and acute painful enlargement. In children, the spleen is the most common site. Massive splenic enlargement may result in severe anaemia, circulatory collapse and death. Recurrent sickling in the spleen in childhood results in infarction and adults may have no functional spleen. In adults, the liver may undergo sequestration with severe pain due to capsular stretching. Priapism is a complication seen in affected men.
Sickle-cell anaemia Clinical features (continued) • Aplastic crisis. Infection with human parvovirus B19 results in a severe but self-limiting red cell aplasia.This produces a very low haemoglobin, which may cause heart failure. Unlike in all other sickle crises, the reticulocyte count is low.
Clinical and laboratory features of sickle-cell disease.
Sickle-cell anaemia InvestigationsPatients with sickle-cell disease have a compensated anaemia, usually around 60–80 g/L. The blood film shows sickle cells, target cells and features of hyposplenism. A reticulocytosis is present. The presence of HbS can be demonstrated by exposing red cells to a reducing agent such as sodium dithionite; HbA gives a clear solution, whereas HbS polymerises to produce a turbid solution.This forms the basis of emergency screening tests before surgery in appropriate ethnic groups but cannot distinguish between sickle-cell trait and disease. The definitive diagnosis requires haemoglobin electrophoresis to demonstrate the absence of HbA, 2–20% HbF and the predominance of HbS. Both parents of the affected individual will have sickle-cell trait.Sickle-cell anaemia Management All patients with sickle-cell disease should receive prophylaxis with daily folic acid, and penicillin V to protect against pneumococcal infection, which may be lethal in the presence of hyposplenism. These patients should be vaccinated against pneumococcus, meningococcus, Haemophilus influenzae B, hepatitis B and seasonal influenza. Vaso-occlusive crises are managed by aggressive rehydration, oxygen therapy, adequate analgesia (which often requires opiates) and antibiotics.
Sickle-cell anaemia Management (continued) Transfusion should be with fully genotyped blood wherever possible. Simple top-up transfusion may be used in a sequestration or aplastic crisis. A regular transfusion programme to suppress HbS production and maintain the HbS level below 30% may be indicated in patients with recurrent severe complications, such as cerebrovascular accidents in children or chest syndromes in adults. Exchange transfusion, in which a patient is simultaneously venesected and transfused to replace HbS with HbA used in life-threatening crises or to prepare patients for surgery.
Sickle-cell anaemia Management (continued) A high HbF level inhibits polymerisation of HbS and reduces sickling. Patients with sickle-cell disease and high HbF levels have a mild clinical course with few crises. Some agents are able to increase synthesis of HbF and this has been used to reduce the frequency of severe crises. The oral cytotoxic agent hydroxycarbamide has been shown to have clinical benefit with acceptable side effects in children and adults who have recurrent severe crises. Allogeneic stem cell transplants from HLA-matched siblings appears to be potentially curative .
Sickle-cell anaemia Prognosis In Africa, few children with sickle-cell anaemia survive to adult life without medical attention. Even with standard medical care, approximately 15% die by the age of 20 years and 50% by the age of 40 years.
Other abnormal haemoglobins Another beta chain haemoglobinopathy, haemoglobin C (HbC) disease, is clinically silent but associated with microcytosis and target cells on the blood film. Compound heterozygotes inheriting one HbS gene and one HbC gene from their parents have haemoglobin SC disease, which behaves like a mild form of sickle-cell disease. SC disease is associated with a reduced frequency of crises but is not uncommonly linked with complications in pregnancy and retinopathy.
The thalassaemiasThalassaemia is an inherited impairment of haemoglobin production, in which there is partial or complete failure to synthesise a specific type of globin chain. In alphathalassaemia, disruption of one or both alleles on chromosome 16 may occur, with production of some or no alpha globin chains. In beta-thalassaemia, defective production usually results from disabling point mutations causing no (β0) or reduced (β–) beta chain production.
Beta- thalassaemiaFailure to synthesise beta chains (beta- thalassaemia) isthe most common type of thalassaemia, most prevalentin the Mediterranean area. Heterozygotes have thalassaemia minor, a condition in which there is usually mild anaemia and little or no clinical disability, which may be detected only when iron therapy for a mild microcytic anaemia fails. Homozygotes (thalassaemia major) either are unable to synthesise haemoglobin A or, at best, produce very little; after the first 4–6 months of life, they develop profound hypochromic anaemia.
The thalassaemias Management and prevention Cure is now a possibility for selected children, with allogeneic haematopoietic stem cell transplantation. It is possible to identify a fetus with homozygous beta-thalassaemia by obtaining chorionic villous material for DNA analysis sufficiently early in pregnancy to allow termination. This examination is only appropriate if both parents are known to be carriers (beta-thalassaemia minor) and will accept a termination.
Diagnostic features of beta-thalassaemia
Treatment of beta-thalassaemia major Problem Erythropoietic failure Allogeneic HSCT from HLA-compatible sibling Transfusion to maintain Hb > 100 g/L Folic acid 5 mg daily Iron overload Iron therapy contraindicated Iron chelation therapy Splenomegaly causing mechanical problems, excessive transfusion needs Splenectomy.
(HLA = human leucocyte antigen; HSCT = haematopoietic stem cell transplantation)
Alpha- thalassaemiaReduced or absent alpha chain synthesis is common inSoutheast Asia. There are two alpha gene loci on chromosome 16 and therefore each individual carries fouralpha gene alleles.• If one is deleted, there is no clinical effect.• If two are deleted, there may be a mild hypochromic anaemia.• If three are deleted, the patient has haemoglobin Hdisease.• If all four are deleted, the baby is stillborn (hydropsfetalis).
Haemoglobin H is a beta-chain tetramer, formed from the excess of beta chains, which is functionally useless, so that patients rely on their low levels of HbA for oxygen transport. Treatment of haemoglobin H disease is similar to that of beta-thalassaemia of intermediate severity, involving folic acid supplementation, transfusion if required and avoidance of iron therapy.