Neutrophil granulocytes

Neutrophils are the most numerous leukocytes in the blood of the healthy adult. The nucleus of the neutrophil granulocyte is characteristically segmented into up to five lobes and the nuclear chromatin stains densely (Fig. 23.1). The abundant cytoplasm stains pink and contains characteristic granules. Within the granules are enzymes, including myeloperoxidase, alkaline phosphatase and lysozyme. Neutrophils have a scavenging function and are of particular importance in defence against bacterial infection.

Neutrophil precursors and neutrophils spend 14 days in the bone marrow, whereas the half-life of neutrophils in the blood is only 6–9 hours. Peripheral blood counts therefore measure less than 10% of the total body neutrophils. Within the circulation the cells move between a circulating and a ‘marginating’ pool, margination being attachment to vascular endothelial cells. To perform their scavenging function, granulocytes irreversibly enter the tissues by penetrating endothelial cells modified by inflammatory mediators. Cytokine-stimulated endothelial cells present adhesion molecules which interact with neutrophils and facilitate their passage: one such is ICAM-1 (intercellular adhesion molecule 1).

Lymphocytes

The peripheral blood lymphocytes are small leukocytes with a round or only slightly indented nucleus and scanty sky-blue-staining cytoplasm which may contain an occasional pink- or red-staining granule. Circulating B- and T-cells are not distinguishable by morphology alone. Immunological staining shows that in health approximately 70% of circulating small lymphocytes are T-cells and 30% B-cells.

A small proportion of lymphocytes may be larger with abundant cytoplasm, sometimes referred to as ‘activated’ lymphocytes. These are believed to represent cells that have been stimulated, perhaps by foreign antigen. A more complete description of the classification and role of lymphocytes is to be found in Chapter 9.

Monocytes

Monocytes are the largest blood cells. The nucleus is oval or reniform but not lobed. The abundant cytoplasm stains pale blue and often contains pink granules; vacuoles are often present. The function of monocytes is similar to that of neutrophil granulocytes: they enter the tissues and, as tissue macrophages, are responsible for the phagocytosis and digestion of foreign material and dead tissue.

Eosinophil granulocytes

Eosinophil granulocytes have much larger red-staining granules. They contain enzymes, including a peroxidase. The nucleus is lobulated, but usually only two or three lobes are seen. The eosinophil is important in the mediation of the allergic response and in defence against parasitic infestation.

Basophil granulocytes

Basophil granulocytes are the least frequent leukocytes in normal blood. The granules are large, blue–black and obscure the bilobed nucleus; they contain heparin and histamine. Basophils are closely related to tissue mast cells but their function has not been determined precisely. They appear to be key mediators of immediate hypersensitivity reactions, involving release of histamine.

Quantitative changes

Leukocytosis means an increase in numbers of circulating white blood cells. Depending on the cause, there may be a polymorphonuclear leukocytosis (neutrophilia—increased neutrophil leukocytes), monocytosis, eosinophil leukocytosis (eosinophilia), basophil leukocytosis (basophilia) or lymphocytosis.

Causes of reactive neutrophil leukocytosis include:

Monocytosis may be reactive to:

Eosinophil leukocytosis may be reactive to:

Lymphocytosis is most commonly associated with an infection such as infectious mononucleosis, tuberculosis, etc.

In some disorders the leukocytosis may be extreme (for example 100 × 10⁹/l), particularly in children. There may also be a tendency for immature leukocytes, particularly myelocytes and metamyelocytes, to appear in the peripheral blood. Severe bacterial infection may result in such an extreme reactive picture, which has in the past been referred to as a ‘leukaemoid reaction’ because of the similarity of the blood picture, with immature forms present, to that of chronic myeloid leukaemia. Occasionally, the lymphocyte series may be involved in such an extreme reactive process, especially during childhood viral infection.

A characteristic leukocytosis composed of ‘atypical’ lymphocytes is a feature of infectious mononucleosis (glandular fever). The infection is common in young adults and often manifests as a sore throat with enlarged lymph nodes and spleen and skin rash. It is due to infection with Epstein–Barr (EB) virus and is common between 15 and 25 years of age in developed countries, but occurs in young children in heavily populated developing countries. The major additional features are:

The atypical cells in peripheral blood are recognisable as lymphocytes but are much larger and have abundant cytoplasm and nuclear irregularities (Fig. 23.5). They are probably reactive T-lymphocytes responding to B-lymphocytes containing the virus, are detectable in blood about 7 days after the onset of illness and may persist for 6 weeks or more. Apparently fortuitously, but usefully, antibodies reactive against horse, sheep and ox red cells (heterophile antibodies) typically develop during the second week and may persist for a few months; they are detected in the Paul–Bunnell test or by more convenient commercial screening slide tests such as the ‘Monospot’ test, and are of diagnostic value. A very similar clinical and haematological (but not serological) picture can develop as a result of other infections, especially with human immunodeficiency virus (HIV), cytomegalovirus and toxoplasma.

Fig. 23.5 Atypical mononuclear cells in infectious mononucleosis. These large T-lymphocytes have copious basophilic cytoplasm with irregular cell outline.

All of the above are examples of reactive leukocytosis. Increased white cell counts in peripheral blood, often with immature forms present, are also a typical feature of some primary disorders of the bone marrow, especially leukaemias and myeloproliferative disorders.

A reduction in circulating leukocytes is termed leukopenia. Most important is a deficiency of neutrophil granulocytes— neutropenia. Neutropenia is commonly seen in association with a reduction in other blood cells, that is, as part of a pancytopenia. Important causes of pancytopenia are:

Important causes of selective neutropenia are:

In cyclical forms the neutropenia is temporary and recurrent, often with a periodicity of 3–4 weeks. It is an uncommon condition.

Neutropenia with counts of less than 0.5 × 10⁹/l may result in severe sepsis, especially of the mouth, pharynx (Fig. 23.6) and peri-anal regions, and also in disseminated infection. This clinical picture is now most commonly seen in patients receiving drug or irradiation therapy for malignant disorders.

Fig. 23.6 Oral infection with Candida albicans (‘thrush’) in a neutropenic patient.

Qualitative changes

Qualitative leukocyte changes are less important than quantitative abnormalities. Defects of phagocytic cell function resulting in an increased tendency to bacterial infection are recognised, particularly as acquired defects after splenectomy, in leukaemic disorders and due to corticosteroid therapy. Congenital abnormalities of leukocyte function are uncommon. ‘Atypical’ lymphocytes in infectious mononucleosis have been described earlier. Other abnormalities of neutrophil morphology are also recognised (Fig. 23.7).

Fig. 23.7 Abnormalities of neutrophil morphology.

Deficiency of lymphocytes in blood is termed lymphopenia. It is often due to medication with immunosuppressive or cytotoxic drugs, for example. Lymphopenia is an important feature of infection with HIV.

Erythrocyte sedimentation rate

The erythrocyte sedimentation rate (ESR) measures the rate at which red cells sediment by gravity in plasma in 1 hour and is a widely used laboratory test. Increased aggregation and sedimentation occur in the presence of high concentrations of immunoglobulin and fibrinogen. As the latter is an acute phase reactant, the ESR is increased in a wide variety of inflammatory and neoplastic conditions. It is an entirely non-specific test and a normal value for ESR can never be used to exclude the presence of significant disease. Direct measurement of plasma viscosity provides equally useful data and has replaced ESR measurement in some diagnostic laboratories.

Iron metabolism

Iron is an essential requirement. It is also one of the commonest elements present in the Earth’s crust. Excessive iron deposited in the tissues is, however, toxic, causing damage to the myocardium, pancreas and liver in particular (Ch. 16). As the body has no active method for iron excretion, iron status is controlled largely by its absorption; the capacity to absorb iron is, however, limited and any tendency to increased loss of iron, due to haemorrhage, is highly likely to result in a negative iron balance and iron deficiency. These factors explain the high prevalence of iron deficiency.

Normally, at least 60% of the body iron is in the haemoglobin of erythroid cells. Approximately 30% is stored within the reticulo-endothelial system (also known as the mononuclear phagocyte system), especially in the bone marrow, as ferritin and haemosiderin. A small proportion of total body iron is present in other tissues, especially muscle, and iron-containing enzymes. This tissue iron is relatively conserved during states of iron deficiency. Only a small fraction of the total body iron is in transport, attached to the carrier protein transferrin.

Ferritin is a protein–iron complex. The protein, apoferritin, is a shell made up of 22 subunits. The core is composed of ferric oxyhydride. Haemosiderin consists of partially degraded ferritin aggregates. Ferritin is present in all tissues, but especially in the macrophages of the bone marrow and spleen and in hepatocytes. A small amount is detectable in plasma and, as it is derived from the storage pool of body iron, its concentration is thus an accurate indicator of body iron stores. Low serum ferritin concentration is a useful confirmatory test for iron deficiency. However, because ferritin is an acute phase response protein, the concentration in plasma is not a reliable guide to body iron stores in the presence of infection, inflammation and neoplasia. In those situations serum ferritin may be normal or high despite tissue iron depletion.

Ferritin is water soluble and not visible by light microscopy; haemosiderin is insoluble and forms yellow granules. When exposed to potassium ferrocyanide (Perls’ stain) the granules are blue–black. Examination of aspirated bone marrow stained with Perls’ stain can therefore be used to assess body iron stores reliably. When iron stores are normal, haemosiderin is visible, mainly in the reticulo-endothelial cells of the bone marrow. In iron overload, most of the iron is in the form of haemosiderin and can be easily identified.

Transferrin is an iron-binding beta-globulin responsible for iron transport and delivery to receptors on immature erythroid cells. Each molecule of transferrin can bind two atoms of iron, but normally the transferrin is only one-third saturated (thus the serum iron concentration is normally one-third of the total serum iron-binding capacity). Transferrin is reutilised after delivering its iron. A low transferrin saturation is therefore diagnostic of iron deficiency while high levels are a feature of iron overload with deposition of iron in tissues.

In order to maintain iron balance, sufficient iron must be absorbed to replace that lost from the urinary and gastrointestinal tracts as shed cells and in sweat, together with any extra requirements.

Daily iron requirements are:

Thus, requirements vary with circumstances, extra iron being required for growth during childhood, for the fetus and placenta and expansion of maternal red cell mass during pregnancy, and to compensate for menstrual loss of women of child-bearing age.

As a Western diet contains only 10–20 mg of iron per day and only a maximum of one-third of this can be absorbed, excess losses of iron of just a few milligrams will inevitably result in negative iron balance and eventual depletion of iron stores. One millilitre of blood contains 0.5 mg iron. Thus, loss of 10 ml of blood daily will inevitably exceed the capacity to absorb sufficient iron, even from a good diet. This explains the finding of some degree of iron depletion in 25% or more of menstruating women.

Iron absorption takes place in the duodenum and upper jejunum. Haem iron is present in meat and readily absorbed, with little effect from other dietary components. Inorganic iron in vegetables and cereals is mostly trivalent and may be complexed to amino acids and organic acids, from which it must be released and reduced to the divalent state for absorption. HCl produced by the stomach and ascorbic acid in food favour its absorption. In contrast, phosphates and phytates in some foods form precipitates and prevent absorption.

Mechanisms controlling the rate of iron absorption are becoming better understood. Major influences are the total body iron stores and rate of erythropoiesis. Thus, if iron stores are replete a smaller proportion of available iron is absorbed; when erythropoiesis is more active, due to premature red cell destruction for example, extra iron is absorbed even though total stores may be high. This is a feature in thalassaemia, and iron overload may ensue. A major regulator of iron balance is the liver protein hepcidin. Iron loading leads to rapid production of hepcidin by the liver which, in turn, inhibits intestinal iron absorption and movement of iron from stores. This is achieved by hepcidin downregulating the plasma membrane transfer protein ferroportin. Iron is consequently trapped in iron-exporting cells, including duodenal enterocytes. Plasma iron levels subsequently fall. Conversely, low iron levels lead to downregulation of liver hepcidin and increased iron transfer from the gut and iron-exporting cells so that plasma iron levels rise (Fig. 23.14).

Fig. 23.14 The role of hepatic hepcidin in regulating iron stores. Hepcidin binds to ferroportin and internalises it, trapping iron within iron-exporting cells. (IL-6, interleukin 6.)

Mechanisms of iron deficiency

In developed countries, iron deficiency in the non-menstruating and non-pregnant adult most frequently results from chronic blood loss, often from the gastrointestinal tract. As it is possible to lose several millilitres of blood daily into the gut lumen without marked change in appearance of the stool, such blood loss is frequently occult. Iron deficiency anaemia is thus commonly a presenting feature of lesions within the gastrointestinal tract (Fig. 23.15). In clinical practice, when iron deficiency anaemia occurs in the face of a reasonable diet and no excessive menstrual loss it is mandatory to perform a careful assessment of the gastrointestinal tract.

Fig. 23.15 Carcinoma of caecum causing iron deficiency anaemia. An annular carcinoma can be seen as a lesion that causes narrowing of the barium-filled bowel (arrowed) and from which blood loss has occurred.

Causes of iron deficiency are:

More than one factor may operate concurrently. Thus a poor-quality vegetarian diet is highly likely to result in iron deficiency in a menstruating female. In a male or post-menopausal female, failure to ingest or absorb any iron would result in complete depletion of iron stores only after 3 or more years (1 mg/day). Malnutrition or malabsorption is thus rarely the sole cause of iron deficiency, although it may be an important contributory factor.

The microcytic hypochromic anaemia is a late stage in iron deficiency; it does not occur until iron stores are severely depleted. The microcyte results from an extra cell division, in addition to the normal four, during red cell production. Increasing cytoplasmic haemoglobin concentration normally acts as an inhibitor of normoblast division. The failure of haemoglobin synthesis that results from iron deficiency therefore allows extra mitoses to occur, with the production of small erythrocytes. The same mechanism is responsible for the microcytes in thalassaemia, another disorder of haemoglobin synthesis.

Blood and bone marrow changes

The typical blood picture is one of microcytic, hypochromic red cells, with increased anisocytosis and poikilocytosis; elongated ‘pencil’ or ‘cigar’ cells are typically present (Figs 23.3 and 23.16). The proportion of polychromatic cells (or reticulocytes) is low for the degree of anaemia, indicating an inability of the bone marrow to respond due to lack of iron for haemoglobin synthesis. The platelet count is often raised, especially if chronic bleeding is present. The leukocytes are typically normal.

Fig. 23.16 The blood in iron deficiency: microcytic hypochromic anaemia. There is central pallor of the erythrocytes and poikilocytosis with elongated (pencil or cigar) cells.

Occasionally, a mixture of microcytic, hypochromic erythrocytes and macrocytic cells is seen. This is termed a dimorphic picture and occurs in mixed deficiency of iron and folic acid or vitamin B₁₂. The MCV and MCH may be misleadingly normal.

A mixture of microcytic, hypochromic cells and normocytes is present in iron deficiency responding to iron replacement or after transfusion of a subject with iron deficiency anaemia (Fig. 23.17). In the former circumstance, mildly increased polychromasia (and reticulocytosis) may be present.

Fig. 23.17 A dimorphic blood film in iron deficiency anaemia responding to oral iron therapy. Hypochromic microcytes (arrowhead) and normocytes (arrow) are present.

Abnormalities are also present in the bone marrow. The nucleated red cell precursors are small in diameter and the cytoplasm is frequently ragged—micronormoblastic erythropoiesis. Staining for haemosiderin (Perls’ stain) reveals its absence from macrophages and normoblasts.

Important biochemical changes in the blood are a fall in serum iron and increase in total iron-binding capacity (representing a compensatory increased transferrin concentration). Saturation of iron-binding capacity is thus reduced to 10% or less, from the normal 33%. The serum ferritin is generally markedly reduced, corresponding to severely depleted body iron content. This situation contrasts with the anaemia associated with chronic inflammatory disease or neoplasia (‘anaemia of chronic disorders’), where red cells are often normocytic but may be mildly microcytic. The serum iron may also be low but this is misleading as iron stores are normal. In contrast to iron deficiency, total iron-binding capacity is usually reduced in anaemia of chronic disorders, and serum ferritin is often raised due to the presence of inflammation or malignancy.

Changes in other organs and tissues

In addition to the manifestations of chronic anaemia, a variety of epithelial changes may be present in chronic iron deficiency:

The cause of these phenomena is unknown. Angular cheilitis (Fig. 23.18), painful fissuring of the mouth corners, is common but not specific: it occurs in dental malocclusion, most often due to poorly fitting dentures. Smooth tongue is also common (Fig. 23.18). Gastric achlorhydria appears to be an occasional result, as well as a contributory cause, of iron deficiency.

Fig. 23.18 Angular cheilitis and smooth tongue in iron deficiency.

Dysphagia (difficulty in swallowing) due to the presence of a web or fold of mucosa in the post-cricoid region is an uncommon association of iron deficiency. The combination has been termed Paterson–Kelly or Plummer–Vinson syndrome and is important mainly because the mucosal abnormality is premalignant, carcinoma occasionally developing at the site.

Koilonychia (spoon-shaped nails) of chronic tissue iron depletion is typical but only rarely seen.

The pathological changes of iron deficiency are reversed by adequate replacement therapy by the oral route.

Vitamin B₁₂ metabolism

Vitamin B₁₂ is necessary for DNA synthesis. Deoxyadenosyl-cobalamin is the main form of vitamin B₁₂ in tissues and methylcobalamin is the main form in plasma. These forms differ only in the type of chemical group (deoxyadenosyl or methyl) attached to the cobalt atom which is located at the centre of a corrin ring, to which a nucleotide portion is attached. (The corrin ring is similar to the porphyrin ring of haem.) The vitamin is known to be a coenzyme in the methylation of homocysteine to methionine and also in conversion of methylmalonyl CoA to succinyl CoA. During the former reaction, methylcobalamin loses its methyl group and this is replaced from methyltetrahydrofolic acid, the principal form of folic acid in plasma. The tetrahydrofolic acid is essential for the generation of deoxythymidine monophosphate, a precursor of DNA. Metabolism of vitamin B₁₂ and of folate are thus closely related and essential for nucleic acid production (Fig. 23.19).

Fig. 23.19 The roles of vitamin B₁₂ and folate in DNA synthesis.

Vitamin B₁₂ is present in foods of animal origin. It cannot be synthesised by higher animals but is produced by micro-organisms. Animals obtain the vitamin from bacterially contaminated foods. Cereals, fruit and vegetable foods contain no vitamin B₁₂ unless they have undergone bacterial contamination. Milk and eggs contain sufficient vitamin B₁₂ for human needs (1–2 mg daily) and thus dietary deficiency can occur only if a strictly vegetarian (vegan) diet is consumed. Nutritional vitamin B₁₂ deficiency (in contrast to dietary folate deficiency) is thus rarely encountered.

Vitamin B₁₂ released from food in the stomach becomes bound to a glycoprotein produced by gastric parietal cells —intrinsic factor. The complex of cobalamin and intrinsic factor binds to receptors on the mucosal cells of the terminal ileum, where vitamin B₁₂ is absorbed and intrinsic factor remains in the lumen of the bowel. In the absence of intrinsic factor, cobalamin cannot be absorbed.

Vitamin B₁₂ is transported to the tissues attached to a plasma-binding protein—transcobalamin II. Another transcobalamin (transcobalamin I), synthesised by neutrophil granulocytes, binds the greater proportion of plasma vitamin B₁₂ but does not liberate it efficiently. The function of transcobalamin I-bound vitamin B₁₂ is unknown.

Body stores of vitamin B₁₂ amount only to some 2–3mg. However, only 1μg daily is required for normal DNA synthesis. Several years must therefore have elapsed before a deficiency state develops, even in the absence of absorption of the vitamin. Twenty micrograms or more per day is available in a mixed diet.

Mechanisms of vitamin B₁₂ deficiency

Causes of vitamin B₁₂ deficiency are:

Addisonian pernicious anaemia accounts for by far the majority of cases of megaloblastic anaemia due to deficiency of vitamin B₁₂. Other causes are uncommon. Some cases occur after gastric resection, usually total gastrectomy. Blind loops of bowel have previously been the result of gastric surgery and this is now a rare cause of vitamin B₁₂ deficiency, with improvements in the medical and surgical management of gastric and duodenal disease.

Addisonian pernicious anaemia is a common disorder in which chronic atrophic gastritis and failure of intrinsic factor synthesis lead to malabsorption of vitamin B₁₂ and, after several years, the development of megaloblastic anaemia. Untreated, this was severe and eventually fatal; the condition was indeed ‘pernicious’ but is now easily corrected by injections of vitamin B₁₂.

Pernicious anaemia is due to an autoimmune process, resulting in atrophy of the chief and parietal glands of the stomach, with consequent failure of acid and intrinsic factor production. An auto-antibody to parietal cells is present in the serum in the majority of cases, but is not specific to this disorder. Antibodies to intrinsic factor are present in 50–70% of cases and much more specific to pernicious anaemia. Absence of anti-intrinsic factor antibodies does not exclude the diagnosis of pernicious anaemia; however, in cases where it is not identified, malabsorption of vitamin B12 can be detected by the Schilling test which measures absorption of an oral dose both in isolation and combined with intrinsic factor. Malabsorption that is corrected by the addition of intrinsic factor is due to pernicious anaemia; malabsorption that is not corrected is due to another cause.

The disease is rather more common in females and rarely presents before 30 years of age, although an uncommon childhood form is occasionally seen. The patient may have another autoimmune disorder such as thyroid disease or vitiligo. There is an association with blue eyes and premature greying of hair.

Blood and bone marrow changes

In contrast to iron deficiency, the defect in DNA synthesis affects all cell lines, and pancytopenia is frequently present. The MCV is high, and oval macrocytes are visible on the blood film (Figs 23.3 and 23.20). In megaloblastic anaemia, a reduction in the number of mitoses during red cell development, due to impaired DNA synthesis with normal RNA and protein synthesis, results in the production of macrocytes. The degree of polychromasia on the blood film is not appropriate to the severity of anaemia, because the marrow is unable to respond.

Fig. 23.20 A macrocytic blood film in megaloblastic anaemia. Oval macrocytes and neutrophil nuclear hypersegmentation (Fig. 23.7) are typical.

A proportion of neutrophil leukocytes have exaggerated lobulation of the nucleus and are often large (neutrophil hypersegmentation). Rarely, the blood picture is leukoerythroblastic.

The bone marrow is hypercellular and the stained smears reveal the characteristic megaloblastic change of the developing red cells (Fig. 23.21): the red cells are larger than normal at each stage of development; nuclear chromatin has a very open appearance, with little condensation, and nuclear development lags behind that of the cytoplasm; thus well-haemoglobinised cells with an immature nucleus are a feature. Multilobed polymorphonuclear leukocytes may be seen, as well as particularly large metamyelocytes and band cells. Megakaryocytes may also appear abnormal.

Fig. 23.21 Bone marrow appearances in megaloblastic anaemia from a vitamin B₁₂-deficient patient. The megaloblasts are extremely large red cell precursors and the nucleus has a very open, speckled pattern. Although some of the megaloblasts are well haemoglobinised, the nucleus is still present, suggesting nuclear/cytoplasmic developmental asynchrony (arrow). The more mature non-nucleated red cells are also large and oval in shape—oval macrocytes (arrowhead).

Biochemical abnormalities detectable in the serum include unconjugated hyperbilirubinaemia and increased concentration of lactic dehydrogenase. These changes are due to increased cell breakdown within the marrow, called ineffective erythropoiesis, and the premature removal of macrocytes, in the reticulo-endothelial system. The serum concentration of vitamin B₁₂ is reduced.

Changes in other organs and tissues

Lesions of the nervous system are a frequent feature of vitamin B₁₂ deficiency from any cause. Myelin degeneration of the posterior and lateral columns of the spinal cord is typical and often associated with a peripheral neuropathy affecting sensory neurones. This subacute combined degeneration of the cord causes spasticity, reduced coordination and impaired sensation in the lower limbs and may be present despite normal haemoglobin levels, although the megaloblastic erythropoiesis is always detectable. Conversely, extreme megaloblastic change and profound pancytopenia may be present without evidence of damage to the nervous system from vitamin B₁₂ deficiency. Optic atrophy and cerebral changes resulting in psychiatric disease are less common accompaniments of deficiency of vitamin B₁₂. The cause may be failure of synthesis of S-adenosyl methionine necessary for myelin formation. Deficiency of folate is not generally associated with the neurological features seen in cobalamin deficiency, although psychiatric abnormalities may occur.

Mucosal abnormalities may be present. Atrophic glossitis is a common feature. In pernicious anaemia there is atrophy of the glands of the gastric body affecting chief cells and parietal cells; there is replacement by mucus-secreting goblet cells. The intestinal epithelial cells are often larger than normal, reflecting megaloblastic change akin to that in the bone marrow.

In addition to the above, changes may be present in the heart and elsewhere due to the chronic hypoxia of severe anaemia. Cardiomyopathy is a particularly important feature; transfusion is tolerated badly due to volume overload and may result in fatal cardiac failure.

The clinical features of B₁₂ deficiency are explained by the pathology, although it is unusual for all features to be present together:

Treatment is by parenteral (intramuscular) administration of vitamin B₁₂. Oral replacement is ineffective in pernicious anaemia due to the deficiency of intrinsic factor.

The haematological abnormalities are completely reversed by vitamin B₁₂ replacement; however, the neuropathology and associated clinical features may only be partly corrected. The gastric atrophy and achlorhydria are primary features in pernicious anaemia, not secondary to the deficiency state, and as such do not reverse on treatment of the deficiency. There is a life-long slightly increased risk of carcinoma of the stomach.

The haematological response is manifested by a marked increase in the reticulocyte count 2–3 days after administration of vitamin B₁₂ and maximal at 7 days; the rise is proportional to the severity of the anaemia. White cell and platelet counts recover within several days and haemoglobin increases at about 10 g/l each week, with an accompanying fall in the MCV to normal values. Erythropoiesis is already normoblastic within 48 hours of starting replacement therapy.

Folate metabolism

Folates are required for DNA synthesis. Folate poly-glutamates (pteroylglutamic acid with extra glutamic acid residues) are the main intracellular forms. However, all dietary folates are metabolised to the monoglutamate methyltetrahydrofolate during absorption from the gut and are transported in this form. Folates are necessary for single carbon unit transfer reactions in amino acid interconversions, in purine synthesis and, crucially, in the thymidylate synthetase reaction.

Humans cannot synthesise folates de novo. Vegetables and fruits are especially rich in folates as polyglutamate conjugates, but most foods contain some folate. Absorption occurs in the proximal jejunum. Dietary polyglutamates are, however, very sensitive to heat, and cooking can markedly deplete foods of their available folate.

Body stores of folate, mainly in the liver, are modest, amounting to some 10mg. As up to 200μg is required daily, a deficiency state can develop within weeks, in contrast to deficiency of vitamin B₁₂. Furthermore, folate requirements are markedly increased in pregnancy and in some diseases associated with increased cell turnover, such as chronic haemolysis.

Mechanisms of folic acid deficiency

Causes of folate deficiency are:

Whereas malnutrition is an unusual cause of deficiency of vitamin B₁₂, it is the most common mechanism of folate deficiency. It is most prevalent in the elderly. Overcooking of food and lack of fresh foods contribute.

During pregnancy, folate and iron deficiency may occur if no supplements are given. In contrast, vitamin B₁₂ deficiency is almost unknown, as fertility is impaired in vitamin B₁₂ deficiency and the commonest cause, pernicious anaemia, is a disease of late middle age and after.

In some disorders the folate deficiency is likely to be multifactorial, as in malignant disease, where lack of appetite with resultant malnutrition may aggravate folate deficiency secondary to increased utilisation of folate by the malignant tissues.

Phenytoin and phenobarbital used long term as anticonvulsants probably impair folate absorption and may interfere with folate metabolism.

Some anti-cancer drugs act as folic acid antagonists. Methotrexate inhibits the enzyme dihydrofolate reductase, thus depleting tetrahydrofolate. The antimalarial pyrimethamine acts similarly. Trimethoprim acts as a folate inhibitor in bacteria but is ineffective as an inhibitor in humans.

Blood and bone marrow

In folic acid deficiency, blood and bone marrow changes are indistinguishable from those in vitamin B₁₂ deficiency. The concentration of folic acid in serum and erythrocytes (red cell folate) is reduced.

Oral folic acid supplements result in a complete reversal of the pathological features. Even in malabsorption states, sufficient folate can be absorbed from pharmacological doses. The time course of the response is identical to that in vitamin B₁₂ deficiency.

Contrasting features of vitamin B₁₂ and folate deficiency are listed in Table 23.5.

Table 23.5 Comparison of features of vitamin B₁₂ and folic acid deficiency states

Megaloblastic anaemia is the result of deficiency of vitamin B₁₂ or folate in the vast majority of instances. However, other causes include drugs and congenital defects. Prolonged anaesthesia with N₂O causes inactivation of vitamin B₁₂ by oxidising the cobalt moiety and has resulted in pancytopenia with megaloblastic erythropoiesis. Cases of congenital deficiency of enzymes involved in cobalamin or folate metabolism or purine or pyrimidine synthesis are extremely rare. However, several anti-neoplastic drugs act by inhibition of synthesis of purine or pyrimidine (hydroxyurea, cytosine arabinoside) and can cause reversible marrow pathology similar to megaloblastic haemopoiesis.

Glucose-6-phosphate dehydrogenase deficiency

Deficiency or defect of glucose-6-phosphate dehydrogenase (G6PD) results in impaired reduction of glutathione. Reduced glutathione protects haemoglobin and red cell membrane from oxidative damage. Inherited G6PD deficiency is an uncommon cause of anaemia in the UK but is among the most common genetic disorders worldwide. It is a sex-linked disorder: female heterozygotes are usually asymptomatic and may have some protection from falciparum malaria; this probably explains the high prevalence of the disorder in many parts of the world.

The common isoenzymes are traditionally designated ‘type B’, the most common, ‘type A’ and ‘type A-minus’, found among American blacks (30% and 11% respectively). Type A differs from type B by a single amino acid substitution and is functionally normal. Type A-minus has an additional amino acid substitution resulting in decreased red cell enzyme activity and disease. Typically there is a tendency to the development of an acute haemolytic episode associated with the ingestion of an oxidant drug (for example some antimalarials and antibiotics) and with other stresses such as surgery or infection. Clinically, a self-limiting episode of anaemia and jaundice develops. Treatment centres on avoidance of exposure to known oxidant drugs.

A further variant is found in Mediterranean populations and is associated with the acute haemolytic tendency known as favism, where ingestion of the fava (broad) bean results in acute haemolysis. The responsible oxidant compound has not yet been identified. Again, oxidant drugs, surgical stress and infections may also lead to haemolysis.

Many other less common genetic variants have been recognised. Some result in a more chronic haemolytic state or neonatal jaundice due to haemolysis.

The blood picture during haemolytic crisis includes increased poikilocytosis with contracted red cells, ‘bite’ cells and ‘blister’ cells (poikilocytes with bite-shaped defects or surface blebs). Oxidised, denatured haemoglobin is seen as red cell inclusions (Heinz bodies) attached to the cell membrane, when blood is stained supravitally as in the reticulocyte preparation. Haemolysis is generally self-limiting because of the rapid outpouring of new red cells, with higher G6PD content, from the marrow in response to the falling haemoglobin. The blood picture is normal between haemolytic episodes.

Treatment consists essentially of avoidance of known precipitating factors for haemolysis. Health is generally good between haemolytic episodes.

Pyruvate kinase deficiency

Pyruvate kinase (PK) deficiency is an autosomal recessive disorder which results in congenital chronic haemolytic anaemia. The blood film has increased poikilocytosis. The chronic anaemia is associated with increased erythrocyte 2,3-BPG because of the site of the metabolic block. This situation results in reduced oxygen affinity of haemoglobin and increased oxygen delivery to the tissues; the anaemia is thus less symptomatic than would be expected from its severity. No specific treatment is available.

Sickle cell disease

Due to homozygous inheritance of a gene coding for a haemoglobin variant that becomes crystalline at low oxygen tensions

Characterised by episodes of tissue infarction and chronic haemolysis

The heterozygous state (sickle cell trait) is associated with normal full blood count and no symptoms

Substitution of valine for glutamic acid in position 6 in the beta chain of globin results in a haemoglobin (HbS) that undergoes aggregation and polymerisation at low oxygen tensions. In the homozygote for sickle cell disease, where the majority of the haemoglobin content of the erythrocytes is HbS, this results in distortion of the red cells, which acquire a sickle shape. The consequence of this distortion and the predominant features of sickle cell disease are a chronic haemolytic anaemia and microvascular occlusion, causing ischaemic tissue damage. The results of the latter dominate the clinical picture.

The gene for HbS is common in the West and Central African populations, the Mediterranean, Middle East and some parts of the Indian subcontinent. Carriage of the gene may confer some protection against falciparum malaria.

The gene is carried by 8% of black Americans and 30% of black Africans. The heterozygous state, or sickle cell trait, results in less than 40% HbS, the remainder being mostly normal HbA. Two major bands are therefore present on electrophoresis of haemoglobin: one corresponding to HbS and one to HbA. The carrier is clinically and haematologically essentially normal, sickling occurring only very uncommonly and only under conditions of severe hypoxia. Haematuria is an occasional feature, due to renal papillary necrosis from focal sickling in the renal medulla. Hypoxic sickling in heterozygotes is an avoidable risk of general anaesthesia.

In the homozygote the haemoglobin concentration is low (70–90g/l). Sickle cells and target cells are present on the blood film, as are features of hyposplenism in the adult (Fig. 23.25). (Splenomegaly due to chronic haemolysis is present during childhood but the spleen shrinks progressively due to microvascular occlusion and infarction.) Even in infancy children with sickle cell disease are functionally hyposplenic with an increased risk of capsulate organism infection. The bone marrow is hyperplastic with erythroid hyperplasia. Extramedullary erythropoiesis in the liver and, occasionally, other sites is a minor feature. Pathological changes in other organs result from the effects of local ischaemia (Fig. 23.26). Haemoglobin electrophoresis reveals a characteristic single band of HbS.

Fig. 23.25 Sickle cell disease (homozygous).

Fig. 23.26 The pathogenesis and clinical consequences of sickle cell disease.

Clinical features. These are predictable from the above. There is anaemia and jaundice from infancy. Sickle ‘crises’ of various clinical types occur from an early age. Vascular occlusion with resultant ischaemia causes severe pain, often in the long bones, abdomen or chest. Ischaemic stroke is common. There is convincing evidence that transcranial arterial Doppler ultrasonography can identify children at increased risk of stroke manifested by increased Doppler flow rates. Introduction of an exchange red cell transfusion programme can reduce this risk. Acute sequestration of sickle cells in the liver or (in children) spleen may cause pain and acute exacerbation of anaemia. Between episodes of crisis, health may be good. Cholecystitis, due to the presence of pigment stones, is a frequent occurrence. As in pyruvate kinase deficiency, oxygen affinity of the haemoglobin is low and symptoms of anaemia mild, due to the relatively enhanced O₂ delivery to tissues. Premature death, often from respiratory complications, may occur in early middle age, but longer survival is a feature in some populations.

Treatment. This is essentially conservative, with avoidance of factors known to precipitate crises, especially hypoxia, and provision of warmth and rehydration during crises. Pregnancy may be complicated by an increased tendency to acute sickle crises. Use of vasoconstrictor drugs is contraindicated—this is an issue when local anaesthetic containing adrenaline (epinephrine) is being considered. Exchange transfusion of red cells to reduce the proportion of cells capable of sickling is a useful treatment in severe exacerbations such as acute chest syndrome where lung tissue is often sequestered following the development of infection. In sickle cell disease, a relatively high proportion of HbF in erythrocytes is associated with a less severe clinical course. Hydroxyurea is a drug used in chemotherapy of some malignancies that increases HbF. It reduces the tendency to acute sickle crises in some patients with sickle cell disease.

Haemoglobin C, D and E. These are also the result of point mutations in the beta chain gene. In the homozygous state they produce mild chronic haemolysis with splenomegaly but without the occlusive manifestations of sickle cell disease. They are commonly found in West Africa, India and South-East Asia respectively. Due to their geographical distribution, the gene for HbC is often inherited with that for HbS. HbS-C disease behaves as a mild sickle disease with a particular tendency to venous thrombosis.

Thalassaemias

Due to abnormalities of (alpha or beta) globin chain synthesis

Characterised by a microcytic, hypochromic blood picture

Beta-thalassaemia major results in severe anaemia from infancy, splenomegaly, marrow expansion with bony deformities and premature death

Beta-thalassaemia minor is clinically mild

Alpha-thalassaemias include disorders resulting in intra-uterine death from severe anaemia and heart failure and those producing clinically insignificant disease

In contrast to the abnormal haemoglobin states described above, where a structurally abnormal globin is synthesised but at a normal rate, in thalassaemia the globin chains are of normal composition, but the rate at which one of the globin chains (alpha or beta) is synthesised is reduced. In alpha-thalassaemia the alpha globin chain synthesis is so affected; in beta-thalassaemia the beta chain is affected. Accumulation of an excess of the unaffected globin chains results in damage to the developing and mature erythrocytes.

Again, in contrast to ‘variant haemoglobin’ conditions (such as sickle cell disease) where point mutations affecting coding regions underlie the disorders, in thalassaemias the genetic lesions are of a regulatory nature, affecting the normal expression of the globin structural genes.

Each chromosome 16 has a pair of alpha globin genes, thus each cell has four genes coding for alpha globin, all of them functional. The genes for beta globin, as well as those for gamma and delta, are located in close linkage on chromosome 11.

In the alpha-thalassaemia syndromes there is deletion of all four genes or of three of the four. In alpha-thalassaemia trait, there is deletion of two or only one gene (Table 23.6).

Table 23.6 The alpha-thalassaemia disorders

More than 200 genetic defects responsible for beta-thalassaemia have now been described, predominantly point mutations, in contrast to the deletions that cause much alpha-thalassaemia. The type of defect tends to vary between racial groups. Some defects result in an absence of chain synthesis (β0); in others, chain synthesis is severely restricted but present (β+).

Beta-thalassaemia major (Mediterranean or Cooley’s anaemia)

This is a severe disorder due to the inheritance of two genes for beta-thalassaemia—β+/β+, β0/β0 or occasionally β+/β0. The beta-thalassaemia genes are most frequent in Mediterranean countries, the Middle East and parts of Africa and South-East Asia.

The blood picture is that of a severe microcytic, hypochromic anaemia (haemoglobin concentration 30–60g/l) developing from 3 to 6 months of age, when beta-chain production would have normally replaced the great majority of gamma chain production, leading to the dominance of Hb A(α₂β₂) and only 1% residual fetal haemoglobin (α₂γ₂).

In response to the defective haemoglobin synthesis and haemolysis the red bone marrow is dramatically expanded with gross erythroid hyperplasia. As a result, cortical bone is thinned and new bone deposits on the outer aspect, especially in the skull vault, maxilla and frontal facial bones (Fig. 23.27). Cortical thinning and fractures may develop in the long bones, vertebrae and ribs. The spleen is grossly enlarged, with expansion of the reticulo-endothelial elements and extramedullary erythropoiesis. The liver is similarly affected. Iron overload is apparent and often gross.

Fig. 23.27 X-ray appearances of the skull in beta-thalassaemia major. Characteristic ‘hair-on-end’ appearances of the skull vault due to marrow expansion. Normal subject.

Haemoglobin electrophoresis reveals absent or markedly reduced haemoglobin A. Small (normal) amounts of haemoglobin A₂ are present and the remainder of the haemoglobin is F.

Predictably, the clinical features are those of severe anaemia, including growth retardation, and iron overload secondary to red cell transfusion and a tendency to absorb excess iron as a result of the dyserythropoietic state. Secondary iron overload may result in failure of sexual development due to iron deposition in the pituitary and endocrine organs, as well as diabetes mellitus, liver and heart failure. Facial deformities result from the bone changes. Iron chelation therapy to reduce tissue iron is a crucial part of management and has been shown to prolong life when used in trials in thalassaemia major patients. Commonly, it is introduced once serum ferritin has reached 1000mg (approximately 20–25 units of red cells transfused). Until recently iron chelation could only be efficiently delivered by near continuous subcutaneous infusion of the iron-chelating drug desferrioxamine. However, there are now two orally administered iron chelators available:deferiprone and deferasirox. Bone marrow transplantation has been curative.

Acute lymphoblastic leukaemia

ALL is most common between 2 and 4 years of age. It is the commonest cause of cancer death in childhood. Recent data suggest that one of the predisposing genetic mutations for childhood ALL actually occurs in utero and that further genetic mutations in the first years of life lead to the disease.

ALL blasts were described morphologically by the French– American–British (FAB) classification according to their size, nuclear:cytoplasmic ratio and whether or not there is marked cytoplasmic vacuolation, as L1, L2 or L3 cells. Routine use of panels of monoclonal antibodies has shown that the majority of cases of ALL derive from B-cell precursors, and the use of these antibodies can classify ALL according to the degree of maturation along the B-cell pathway, i.e. common ALL and pre-B ALL, which can be of L1 or L2 morphology, or B-ALL which is of L3 morphology and now known as Burkitt’s leukaemia because of its biological similarity to Burkitt’s lymphoma. The WHO classification distinguishes cases as precursor B-lymphoblastic leukaemia/lymphoma and precursor T-lymphoblastic leukaemia/lymphoma. A number of acquired genetic abnormalities are recognised in ALL. These include the occurrence of a Philadelphia chromosome caused by the t(9;22) translocation in 2% of children but 20–30% of adults with ALL, hypodiploid or hyperdiploid (>50) numbers of chromosomes, t(1;19), t(12;21) and rearrangements of the myeloid/lymphoid or mixed lineage leukaemia (MLL) gene on chromosome 11q.

Acute myeloblastic leukaemia

The incidence of AML increases steadily with age. In older people AML is more likely to develop from an existing bone marrow disorder such as myelodysplastic syndrome (MDS), whereas the majority of younger patients present with de novo AML.

The WHO classification of AML distinguishes subtypes based on biological homogeneity and clinical relevance. As such there are four major categories:1.AML with recurrent genetic abnormalities (including t(8;21), t(15;17), deletion 11q and inversion 16) 2.AML with multilineage dysplasia 3.AML, therapy related (including following exposure to alkylating agents or topoisomerase II inhibitors) 4.AML not otherwise categorised (including cases with evidence of some myeloid, monocytic, erythroid or megakaryocytic differentiation).In all cases there must be at least 20% blast cells in the bone marrow for AML to be diagnosed.

Prognosis in AML is related to acquired cytogenetic abnormalities. As acknowledged in the WHO classification, some of these abnormalities occur in specific subtypes of AML. Patients with balanced translocations t(15;17) in acute promyelocytic leukaemia (APL), t(8;21) and inversion of chromosome 16 respond very well to treatment and have a long-term cure rate of 70–80%. Much is now known about the genes altered by these chromosome rearrangements. For example, in APL with t(15;17) the genes rearranged are PML/RARA. The retinoic acid receptor alpha gene (RARA) in its fused form with PML leads both to the development of this leukaemia and its response to pharmacological doses of all-trans-retinoic acid (ATRA), which is now part of standard therapy for this subtype of AML. At the other end of the spectrum are cytogenetic abnormalities that carry a very poor prognosis with cure rates of less than 20%. These include loss of a whole chromosome 5 or 7 or complex multiple cytogenetic abnormalities. These cases occur more commonly in elderly people, following on from MDS or in so-called treatment-related AML after exposure to chemotherapy for previous cancers. A significant majority of patients have no detectable chromosomal abnormality. However, genetic abnormalities are increasingly recognised in this group of patients using molecular techniques. Examples include internal tandem duplication of the FLT3 gene in 30% of cases which carries a poor prognosis, and a mutation in the nucleophosmin gene which leads to the aberrant location of the protein in the cytoplasm rather than the nucleolus and which carries a relatively favourable prognosis.

Treatment

Treatment of acute leukaemia is directed by individual prognostic assessment. The aim is to offer curative treatment where possible and minimise the long-term complications in groups of patients who have a high cure rate, while intensifying the treatment or using new modes of treatment in those groups of patients who presently do badly. Therefore, as an example of this principle, children and young adults with good prognosis ALL or AML will not be routinely offered bone marrow transplantation, while those with poor prognosis disease may be offered bone marrow transplantation as part of initial treatment. Treatment is by chemotherapeutic agents in combination to clear the blood, bone marrow and other sites of leukaemic blasts as far as is possible. The first one or two courses of treatment are aimed at producing a state of remission, in which the blood counts are normal and there are less than 5% blast cells in the bone marrow as identified by light microscopy. However, light microscopy is not very sensitive and in remission there can still be in the order of 109 leukaemic cells in the marrow. Further courses of chemotherapy are given to consolidate the remission and reduce this leukaemic burden further. In poor prognostic disease it seems chemotherapy alone cannot overcome the leukaemic burden and the additional allogeneic immune attack provided by a stem cell transplant is required to eradicate the disease. This attack is called graft-versus-leukaemia and is mediated by the donor’s engrafted T-lymphocytes. New approaches to leukaemia management include more accurate monitoring of minimal residual disease (disease that cannot be detected by conventional microscopy) by PCR or flow cytometry and acting on the results of such tests. This has been particularly successful in childhood ALL and in APL. New modes of therapy, such as the addition of antibodies targeted against the leukaemia cells, e.g. calicheamicin bound to anti-CD33 (gemtuzumab ozogamicin) in AML, and techniques to harness the graft-versus-leukaemia effect whilst reducing the toxicity of the transplant procedure by using intense T-cell immunosuppression and less myelosuppression (so-called reduced intensity conditioned allografts) are showing promise.

Intensive support by transfusion of blood products and use of antibacterial and antifungal agents is necessary to support the patient during the treatment while bone marrow function is suppressed. Survival is months or a few years in adults, with an increasing proportion of long-term survivors with advances in therapy. The outlook is much better in childhood ALL and in adults with good prognosis AML, where significant cure rates are now achieved.

Blood and bone marrow changes

Leukocytosis is a uniform feature, with occasional cell counts in excess of 300 × 109/l. The cell picture in the blood can superficially resemble that in a bone marrow aspirate, with myelocytes, promyelocytes, myeloblasts and normoblasts present as well as large numbers of band cells and mature polymorphonuclear granulocytes (Fig. 23.37). Basophilia is common. Platelets are increased (sometimes over 1000 ×109/l), normal or reduced. A normochromic anaemia is often present.

Fig. 23.37 The blood in chronic myeloid leukaemia. Myelocytes (arrows) and metamyelocytes (arrowheads) enter the circulation.

The leukocytes are abnormal, as exemplified by an absence or severe reduction of their content of alkaline phosphatase, a feature unique to CML and of diagnostic value. Serum vitamin B₁₂ is elevated due to production of binding protein by the granulocyte series.

The bone marrow is hypercellular with marked reduction of fat spaces; granulocytopoiesis predominates. In the acute, terminal phase increased numbers of blast cells become evident in blood and bone marrow, and anaemia and thrombocytopenia are more marked.

Changes in other organs

The spleen is enlarged, often massively, due to infiltration by CML cells (Fig. 23.38); it may fill the abdominal cavity and extend into the pelvis. Areas of infarction are present due to the rapid enlargement outstripping the available blood supply. Hepatomegaly is also frequently present. Infiltration in other organs is an occasional feature. Infection and bleeding are not common in the chronic phase.

Fig. 23.38 Massive splenomegaly in chronic myeloid leukaemia. The palpable margins of the spleen are indicated.

Clinical course

Symptoms may be mild in the chronic phase and are essentially those of anaemia and massive splenomegaly (abdominal fullness and pain from splenic infarction). Rarely, a hyperviscosity state may develop when the white count is greater than 300 × 109/l. In the acute phase the clinical features are those of acute leukaemia.

Treatment

The treatment of BCR-ABL-positive CML has seen a remarkable change in recent times. With detailed understanding of the molecular structure of the causative oncogene BCR-ABL, a drug called imatinib mesilate—trade name Glivec—has been developed. The drug is a small molecule that binds to the ATP-binding site of BCR-ABL and inhibits the function of the protein. In a large randomised clinical trial (IRIS), this oral drug has had very dramatic positive results with >95% of patients achieving complete haematological remission within 12 months and 87% achieving a complete cytogenetic remission by 60 months. After 5 years of follow-up, the estimated overall survival is 89% of patients treated with imatinib. A trial of imatinib with monitoring of response milestones is now regarded as the standard therapy for all patients newly diagnosed with BCR-ABL-positive chronic phase CML. Some patients will fail to meet the response milestones whilst on imatinib and some who do respond subsequently develop resistance to the drug because of mutations in the ATP-binding site or multiple copies of the BCR-ABL gene. Such patients who are young enough, fit enough and have a suitable donor can be cured by an allogeneic stem cell transplant. Newer tyrosine kinase inhibitors with a broader range of cellular targets (dasatinib) or more potent anti-BCR-ABL activity (nilotinib) are being introduced for patients who fail imatinib therapy and for whom stem cell transplantation is not available. Imatinib and the newer tyrosine kinase inhibitors are also more effective in treating patients in blast crises than conventional chemotherapy. It is too early to know if imatinib or its successors will cure patients with CML; however, the introduction of imatinib is a landmark event and marks the proof of principle that a clear understanding of the pathogenesis of a disease at a molecular level can lead to the design of effective targeted treatment. If tyrosine kinase inhibitor therapy is not available, the proliferative features of CML can be controlled with oral hydroxycarbamide and the chronic phase prolonged with the use of alpha-interferon with or without cytosine arabinoside.

Aetiology

CLL is a chronic lymphoproliferative disorder with features similar to those of a low-grade lymphoma but with predominant blood and marrow involvement. During the last few years there has been significant new understanding of the pathology of this disease. The disease process is a relentless accumulation of B-lymphocytes that appear resistant to apoptosis. In the majority of cases it is a considerably less aggressive disorder than are the other leukaemias. This common form of the disease is a disease of the elderly. It is slowly progressive, usually following a predictable clinical course (Fig. 23.39) with a median survival of 25 years and often does not require therapy. In this form of the disease the malignant B-cell has undergone rearrangement of its immunoglobulin genes and the cell has also passed through the germinal centre of the lymph node and been selected for antigen by hypermutation of its rearranged immunoglobulin genes. Such cells therefore are the leukaemic equivalent of memory B-cells. In a proportion of patients the disease is much more aggressive in its behaviour with resistance to chemotherapy and a much shortened median survival of 8–9 years. In these cases the leukaemic B-cell has not been selected for antigen by hypermutation of the immunoglobulin genes and is the leukaemic equivalent of a naive B-cell. In both forms of the disease lymphocytes accumulate in blood, marrow, liver and spleen until the total lymphoid mass is expanded up to 100-fold.

Fig. 23.39 The clinical course and staging of chronic lymphocytic leukaemia. Five stages are recognised in the ‘Rai’ classification.

Blood and bone marrow changes

Leukocytosis is present; up to 99% of nucleated cells are small lymphocytes (Fig. 23.40) of B-cell origin in most instances. The lymphocyte count is between 5 × 109/l and more than 300 × 109/l. The CLL cells tend to fragment during preparation of the blood film, producing many ‘smear cells’ (Fig. 23.40). Anaemia (normocytic) and thrombocytopenia are late developments (Fig. 23.39). However, in up to 10% of cases a secondary autoimmune haemolytic anaemia develops, with reticulocytosis and microspherocytes and a positive direct antiglobulin test result. Serum immunoglobulins are low in the later stages of the disease.

Fig. 23.40 The blood in chronic lymphocytic leukaemia. Numerous small lymphocytes and ‘smear’ cells (arrows) are characteristic.

The bone marrow is hypercellular, with progressive replacement of normal tissue by small lymphocytes, resulting eventually in anaemia and thrombocytopenia.

Advanced stage non-Hodgkin’s lymphoma (NHL) may result in blood and marrow involvement superficially resembling CLL. However, extensive involvement usually occurs late in the course of the disease and the lymphoma cells are morphologically distinct from the lymphocytes of CLL. Immunophenotyping of B-CLL cells shows a distinct pattern of antigen staining, with the leukaemic cells staining positively for the B-cell antigen CD19 and also for CD5 and CD23. This pattern of staining is useful for helping to distinguish CLL from other cases of B-NHL appearing in the blood which lack staining for CD5 and/or CD23. Cases of CLL, as with other leukaemias, have acquired cytogenetic abnormalities within the leukaemic cells. The common ones are deletions of 13q, 11q and 17p (loss of p53) and trisomy 12. As with acute leukaemia, these abnormalities have prognostic significance, with deletion of 11q and of p53 carrying a poorer prognosis.

Changes in other organs

The lymph nodes, liver and spleen are characteristically involved. In nodes and spleen the normal architecture becomes completely effaced by the infiltrate of monomorphic small lymphocytes, and similar cells are present in the portal tracts of the liver. Occasionally, the predominant presentation is with lymph node involvement with little or no evidence of disease in the blood and marrow, when it is termed small cell lymphocytic lymphoma (WHO-CLL/SCLL).

Clinical course and treatment

For the good prognostic form of the disease the clinical course is protracted; it is summarised in Figure 23.39. The protracted course means that many cases are diagnosed as a result of routine blood tests or clinical examination for some other reason. Elderly patients with this form of the disease often die from an unrelated cause. Many of these patients do not require treatment at all or for many years. Treatment is indicated for the development of significant cytopenias, bulky lymphadenopathy or hepatosplenomegaly, or systemic symptoms such as loss of weight or night sweats. Successful first-line treatment options include single agent chlorambucil or fludarabine alone or in combination with cyclophosphamide.

For the aggressive form of the disease occurring in younger patients and in patients with relapsed disease the outlook is less favourable. Treatment should still be initiated only when there are clinical indications, and treatment regimens involving intensive combinations of chemotherapy, such as fludarabine and cyclophosphamide with the anti-CD20 antibody rituximab, CHOP combination chemotherapy, stem cell transplantation, and the anti-CD52 monoclonal antibody alemtuzumab are therapeutic options to be considered.

Patients with autoimmune manifestations including autoimmune haemolysis and immune thrombocytopenic purpura are treated with corticosteroids.