Haematological Findings in Health and Disease

The blood film and count in healthy individuals

The microscopic features of normal blood cells and the normal range for the blood count have been discussed in Chapter 1. In assessing what is ‘normal’ it is necessary to consider the gender, age and ethnic origin of the person being investigated.

Gender

Adult men have a higher normal range for RBC, Hb and PCV/Hct than adult women but women tend to have a somewhat higher WBC and platelet count (see Table 1.2).

Neonates, infants, and children

The blood counts of healthy neonates, infants and children differ greatly from those of healthy adults (see Table 1.3). Neonates have a higher Hb, MCV, WBC, neutrophil count and lymphocyte count than adults. Children in general have a higher lymphocyte count than adults. They tend to have a slightly lower Hb and MCV.

Pregnancy

Physiological variation in the blood count occurs during preg- nancy. The Hb falls, the MCV rises slightly and the WBC and neutrophil count rise. Immature cells (myelocytes and occasion-al promyelocytes) appear in the blood and there may be ‘toxic’granulation and Döhle bodies. 

Ethnic variation 

The blood counts of healthy Africans and Afro-Caribbeans (see Table 1.3) often show a lower white cell and neutrophil count than is usual in Caucasians (see Table 1.2). There is also a tendency to a lower platelet count, particularly in Africans. 

Abnormalities of red cells 

Polycythaemia 

Polycythaemia is an increase in the Hb. It is usually accompa- nied by an increase in the RBC and PCV/Hct. It can be caused by a true increase in the total volume of red cells in the circulation (true polycythaemia) or by a decrease in the total plasma volume (apparent or relative or pseudo-polycythaemia). It is not possible to distinguish true from apparent polycythaemia by a blood film or count. True polycythaemia is caused by overproduction of red cells. Normally red cell production is driven by erythropoietin production in response to a diminished oxygen supply to the kidney. Overproduction of red cells may be an erythropoietin- mediated physiological response to hypoxia or it may be caused by inappropriate secretion of erythropoietin or by mechanisms independent of erythropoietin. Some of the important causes of polycythaemia, classified according to mechanism, are shown in Table 4.1. 

Polycythaemia vera 

Polycythaemia vera, also referred to as polycythaemia rubra vera or primary proliferative polycythaemia, is a myeloproliferative disorder characterized by overproduction of red cells. In many patients there is also overproduction of white cells and platelets. Clinical features are facial plethora and sometimes a moderate degree of splenomegaly. The disease may be complicated by arterial thrombosis and peripheral ischaemia.

Table 4.1  Some  important causes of polycythaemia, classified according to mechanism.

The blood film is ‘packed’. The number of neutrophils, ba- sophils, and platelets may be increased and some giant platelets may be present. Increased numbers of basophils are particularly important in diagnosis since they are not increased in any of the other causes of true or apparent polycythaemia. 

Further steps: Confirm the high Hb on a repeat blood sample. Check history and physical findings. Is the patient hypoxic or a heavy smoker? Check the blood film for neutrophilia, basophilia and giant platelets. If the cause is not obvious, measure red cell mass and plasma volume to check if this is a true or a relative polycythaemia. If it is a true polycythaemia and the patient is not hypoxic, do an ultrasound examination of the abdomen to assess the kidneys and spleen, measure serum erythropoietin concentration and do a trephine biopsy of the bone marrow. 

Anaemia and other disorders of red cell production 

Anaemia is a reduction of the Hb, usually accompanied by a reduction in the RBC and PCV/Hct. A reduction of the RBC, Hb and PCV/Hct can also be factitious, when a blood specimen has been taken from a vein above an intravenous infusion or when there has been poor mixing of the specimen in the laboratory. Laboratory workers must be alert to the possibility of such mis- leading results. Table 4.2 summarizes the causes of anaemia, according to mechanism. Anaemia can also be classified accord- ing to cell size, as microcytic, normocytic or macrocytic. This is useful as it directs investigation to a more limited range of

Table 4.2  Some  important causes of anaemia, classified according to mechanism.

*A rare mechanism of anaemia except that this is one of the causes of the low Hb in sickle cell anaemia.

Table 4.3  Some  causes of anaemia with microcytic, normocytic or macrocytic red cells.

possibilities (Table 4.3). Anaemia can also be classified according to the appearance of the red cells, e.g. as a spherocytic anaemia or as a microangiopathic haemolytic anaemia. The more important causes of anaemia will be discussed, together with other disor- ders of red cell production, in the following pages. 

Megaloblastic anaemia 

Megaloblastic anaemia usually results from a deficiency of either vitamin B12 or folic acid. It can occur at any age but is common- est in the elderly. Clinical features are those of anaemia (e.g. pallor, fatigue, breathlessness) and in addition there may be mild jaundice, inflammation of the tongue (glossitis) and, in the case of vitamin B12 deficiency, neurological complications.

The diagnosis can only be made with certainty from a bone marrow examination, which shows asynchrony between nuclear and cytoplasmic maturation, nuclear maturation being retarded. However, the blood film and count are often so typical that there is little doubt as to the diagnosis and, if further investigations can be done rapidly and provide a definitive diagnosis, bone marrow examination is not necessary. 

The blood film (see Fig. 3.7) shows anaemia, macrocytosis, anisocytosis and poikilocytosis. Macrocytes include both round and oval macrocytes (whereas round macrocytes alone are charac- teristic of macrocytosis for other reasons, e.g. liver disease or ethanol excess). Anisocytosis and poikilocytosis become marked as the anaemia becomes more severe. Poikilocytes include oval cells, teardrop poikilocytes and red cell fragments. When anaemia is severe, there may be some circulating NRBC, which show the nucleocytoplasmic asynchrony of megaloblastic erythropoiesis (Fig. 4.1). Neutrophils are hypersegmented; there is an increase in the mean lobe count, the proportion of five-lobed neutrophils is increased and some neutrophils with six, seven or more lobes may be present. There may also be a few macropolycytes. 

The FBC shows a fall in the Hb and PCV/Hct and a parallel increase in the MCV and MCH. Mean cell haemoglobin concen- tration is normal. There is a marked fall in the RBC. The RDW is increased. When anaemia is very severe the rise in the MCV is not as marked as might be expected. This is because of the

Fig. 4.1  Megaloblastic anaemia showing macrocytosis and  a circulating megaloblast.

concomitant marked anisocytosis and poikilocytosis with many red cell fragments and small poikilocytes. With severe anaemia the RDW is very abnormal and the HDW is also increased. In severe cases the WBC and platelet count are reduced. 

Further steps: Check clinical history and physical findings. Is the diet deficient in vitamin B12 (vegan or very restricted vegetar- ian diet) or folic acid (lack of liver and fresh fruit and vegetables)? Has the patient had a total gastrectomy? Do the history or the physical findings suggest alcohol excess, liver disease or malab- sorption? Is the patient taking a drug known to cause macrocyto- sis? Are there neurological features suggestive of vitamin B12 deficiency (peripheral neuropathy, optic neuropathy, subacute combined degeneration of the spinal cord, dementia)? 

Assay serum vitamin B12 and red cell folate and do liver and thyroid function tests. Consider whether a bone marrow aspirate is needed. If vitamin B12 concentration is reduced, test for anti- bodies to gastric parietal cells and intrinsic factor and consider a Schilling test. If coeliac disease is suspected, test for antibodies to endomysium and gliadin. 

If haemolytic anaemia is possible, examine blood film for polychromasia and specific features of various haemolytic anae- mias, do reticulocyte count and measure serum bilirubin and lactate dehydrogenase. 

Iron deficiency anaemia 

Iron deficiency anaemia occurs when the body’s stores of iron are insufficient to maintain erythropoiesis. It occurs at any age but is commonest in infancy, in menstruating girls and women and during pregnancy. Clinical features are those of anaemia. Severe cases may also have cracking at the corners of the mouth (angu- lar cheilosis), atrophic glossitis and flat or spoon-shaped nails (koilonychia). 

The anaemia that develops is initially normocytic and normo- chromic and subsequently hypochromic and microcytic (Fig. 4.2). There is mild to moderate anisocytosis and poikilocytosis. Poikilocytes often include elliptocytes. The long, thin ellipto- cytes of iron deficiency are sometimes referred to as pencil cells.

Fig. 4.2  Iron  deficiency anaemia showing anisocytosis, anisochromasia and the  presence of poikilocytes including elliptocytes (pencil cells)

Target cells are uncommon and anisochromasia is characteristic, two features that can be useful in making the distinction from thalassaemia trait. The platelet count is sometimes increased. 

The FBC initially shows a fall in the Hb and a rise in the RDW. Subsequently there is a fall in the MCV and the MCH and, when it is measured by a sensitive technique, a fall in the MCHC. 

Further steps: Check history and physical findings. Is the diet adequate (vegetarian diets are often deficient in iron)? Is the patient a rapidly growing child or adolescent or a woman who has had repeated pregnancies? Is there a history of blood loss, e.g. heavy menses (menorrhagia), vomiting blood (haematemesis), passing black stools (melaena—indicative of upper gastrointesti- nal blood loss)? Does the patient take aspirin or other drugs that cause gastric ulceration or have a history of indigestion or diffi- culty swallowing? Is there any bowel dysfunction suggestive of malabsorption or a bowel lesion that could cause occult haemor- rhage? Consider testing for coeliac disease (antiendomysial and antigliadin antibodies). 

Measure serum ferritin (low level confirms iron deficiency) or both serum iron and iron-binding capacity or transferrin concen-tration (a low serum iron does not confirm iron deficiency unless it is accompanied by a raised iron-binding capacity/ transferrin). 

Remember, it is not sufficient to merely confirm a diagnosis of iron deficiency. You must also find the reason. 

Anaemia of chronic disease 

The anaemia of chronic disease (Fig. 4.3) can result from chronic infection, from chronic inflammatory diseases such as rheuma- toid arthritis, or from malignant disease. Clinical features are those of anaemia and of the primary disease. The mechanism of anaemia is reduced delivery of iron from the reticuloendothelial system to the developing erythroblast together with a blunted erythropoietin response to anaemia and a minor degree of short- ening of red cell survival. The anaemia is initially normocytic and normochromic, but, when the condition is chronic and se- vere, hypochromia and microcytosis may be marked. Poikilocy- tosis is not marked. Because of an increased concentration of plasma proteins, rouleaux formation is usually more marked than in iron deficiency anaemia of equivalent severity and the erythrocyte sedimentation rate (ESR) shows a more marked in- crease. Other signs of an inflammatory response such as in- creased background staining, neutrophilia and thrombocytosis occur if the underlying disease is severe. 

Fig. 4.3  Anaemia of chronic disease showing microcytosis, hypochromia and  anisochromasia

It may be impossible to distinguish between iron deficiency anaemia and the anaemia of chronic disease on the basis of the blood count and film, and assessment of iron stores is then needed. 

Further steps: Check the history and physical findings. Is there evidence of infection, an inflammatory condition or a malignant disease? Are there laboratory markers of inflammation such as neutrophilia, an increased ESR, increased C-reactive protein, in- creased serum globulins. Measure ferritin (normal or high) or both serum iron and iron-binding capacity or transferrin (all low). If it is not clear whether the patient has iron deficiency or the anaemia of chronic disease or a combination of the two, consider a bone marrow aspirate to assess iron stores. 

Beta thalassaemia trait 

Beta thalassaemia trait is the abnormality resulting from reduced or absent function of one of the two beta globin genes. The individual is heterozygous for a beta thalassaemia gene, b0 or b+ thalassaemia. The condition occurs in many ethnic groups in- cluding populations from around the Mediterranean and from the Indian sub-continent and South-East Asia, Africans, Afro- Caribbean and Arabs. This is usually an asymptomatic condi- tion, but, since the offspring of two carriers of beta thalassaemia trait may suffer from thalassaemia major, its detection is impor- tant. The diagnosis is most readily suspected from the FBC. The Hb is usually normal or only slightly reduced although it may be lower during pregnancy or intercurrent infection. The MCV and MCH are reduced and the RBC is elevated. The MCHC may be slightly reduced when it is measured by a sensitive instrument. The high RBC is useful in making a distinction from iron defi- ciency since a high RBC is quite uncommon in iron deficiency. The RDW is more often normal in thalassaemia trait than in iron deficiency. The blood film may show only microcytosis and in such cases the diagnosis may not be suspected from the blood film. In other cases (Fig. 4.4) there is also hypochromia and the presence of poikilocytes including target cells. Some patients have promi-

Fig. 4.4  Beta thalassaemia trait showing microcytosis, hypochromia and poikilocytosis. The  poikilocytes include target cells  and  several irregularly contracted cells.

nent basophilic stippling and some have small numbers of irreg- ularly contracted cells. These two features are not expected in iron deficiency and their presence is therefore useful in the differential diagnosis of microcytosis. 

However, since blood film abnormalities may be subtle, diag- nosis is dependent on the blood count and on carrying out a specific test (measurement of haemoglobin A2 concentration) whenever a low MCV and MCH suggest the possibility of this diagnosis. 

Further steps: Check the ethnic origin of the patient. Measure the haemoglobin A2 percentage. A high level generally confirms beta thalassaemia trait. Don’t forget that genetic counselling may be needed. If the red cell indices are typical of thalassaemia trait but the haemoglobin A2 is normal, consider the possibility of alpha thalassaemia trait (see below). Less often, such indices are the result of polycythaemia that has been treated by repeated venesection and has caused iron deficiency—check the clinical history. 

Alpha thalassaemia trait 

Alpha thalassaemia trait is the abnormality resulting from ab- sence or reduced function of either one or two of the four alpha globin genes. It is an asymptomatic condition. It is commonest among Africans and Afro-Caribbeans and in Chinese and South- East Asian populations. Not all cases show an abnormality of the blood film or count. Patients who lack only one of the four alpha globin genes may have mild microcytosis or may be haematolog- ically normal. Patients who lack two alpha globin genes usually have an abnormality very similar to that of beta thalassaemia trait, although basophilic stippling and target cells are less common. 

Further steps: Usually the diagnosis of alpha thalassaemia trait does not matter. However, in certain ethnic groups (Chinese, South-East Asian, Greek, Cypriot, Turkish) there may be a chro- mosome with no alpha genes. This is called a0 thalassaemia and if it is present in both parents it can cause a severe anaemia, incompatible with life, in a fetus. So check the ethnic origin and arrange DNA analysis if the individual could have alpha thalas- saemia trait and if this would be clinically relevant. If the MCH is 26 fl or higher, a0 thalassaemia is very unlikely. Confirmation of the diagnosis of a+ thalassaemia, the milder condition in which only one of the two alpha genes on a chromosome is missing, is not generally necessary. 

Haemoglobin H disease 

Haemoglobin H disease is an inherited condition characterized by a moderately severe anaemia attributable in part to reduced synthesis of haemoglobin and in part to haemolysis. It is caused by deletion of three of the four alpha globin genes (or by muta- tions of alpha globin genes leading to a similar reduction of alpha globin synthesis). The reduced alpha globin production leads to a thalassaemic disorder and to the production of an abnormal haemoglobin, haemoglobin H, which has four beta globin chains and no alpha chains. Haemoglobin H is present as a minor com- ponent, the majority of haemoglobin being haemoglobin A. Hae- moglobin H disease occurs in South-East Asia and around the Mediterranean. Clinical features are anaemia and splenomegaly. The blood film (Fig. 4.5) shows hypochromia and marked mi- crocytosis and anisocytosis. Poikilocytosis is very striking with poikilocytes including red cell fragments, teardrop poikilocytes

Fig. 4.5  Haemoglobin H disease showing moderate hypochromia and marked microcytosis, anisocytosis and  poikilocytosis.

and target cells. There is polychromasia, correlating with an increased reticulocyte count. 

The Hb is moderately reduced (usually 6–10 g/dl). There is marked reduction of the MCV and MCH, reduction of the MCHC and an increased RDW and HDW. 

Further steps: Look for haemoglobin H inclusions by a specific stain and look for haemoglobin H by haemoglobin electrophore- sis or high performance liquid chromatography (HPLC). Check for a raised reticulocyte count. Check the parents—at least one of them should have only two alpha genes and thus should have obvious alpha thalassaemia trait with an MCH less than 26 fl. 

Hyposplenism 

Reduced or absent splenic function is referred to as hyposplen- ism. It can result from congenital absence or surgical removal of the spleen or from loss of splenic function, e.g. because of splenic atrophy or infarction. The blood film (see Fig. 2.28) shows target cells, Howell–Jolly bodies, acanthocytes, occasional spherocytes, occasional Pappenheimer bodies and some giant platelets. The blood count may show increased neutrophils, lymphocytes or platelets. 

A degree of hyposplenism is normal in the neonatal period, particularly in premature neonates. Otherwise hyposplenism is abnormal and its detection is important. Patients may be una- ware that a splenectomy has been carried out in the past, and they and their physicians may therefore be unaware of the con- sequent risk of overwhelming infection and the need for prophy- lactic penicillin and certain vaccinations. Hyposplenism can also provide a diagnostic clue to conditions such as coeliac disease that can be associated with splenic atrophy. 

When splenectomy is carried out in a patient with a haemato- logical abnormality, post-splenectomy features are often modi- fied or aggravated by the effects of the underlying disease. It is also important to note the features of hyposplenism, when present, since this might explain a haematological abnormality such as thrombocytosis or lymphocytosis and avoid unnecessary further investigations. 

Further steps: Check the history. Is the patient known to have had a splenectomy or is there a history of a disease associated with splenic atrophy (coeliac disease or dermatitis herpetiformis) or replacement of functioning splenic tissue (amyloidosis)? Con- sider a CT scan or other imaging technique to confirm unexpect- ed hyposplenism, as this is an important diagnosis. 

Hereditary spherocytosis 

The term hereditary spherocytosis covers a heterogeneous group of inherited red cell membrane disorders characterized by a membrane spectrin deficiency, spherocytic red cells and either haemolytic anaemia or compensated haemolysis. Inheritance is most often autosomal dominant. Cases occur among many eth- nic groups. Clinical features can include anaemia, jaundice and the complications of gallstones. The blood film shows that some, but not usually all, of the red cells lack central pallor (see Fig. 2.12). In patients with anaemia there may be polychromasia. The Hb may be normal or reduced.

Fig. 4.6  Spherocytes, spheroacanthocytes and  red cells  containing

Pappenheimer bodies following splenectomy for hereditary spherocytosis.

MCHC is increased when it is measured by a sensitive method. After splenectomy, spheroacanthocytes, i.e. spherical cells with irregular spicules, may be very prominent (Fig. 4.6). 

Further steps: Check the family history and, if necessary, the blood counts and films of both parents. An osmotic fragility test confirms the present of spherocytes but is not necessary if it is obvious that there are spherocytes. An autoimmune haemolyt- ic anaemia is an important differential diagnosis, so check the direct antiglobulin test (Coombs’ test). If the patient is in hospi- tal, check that there has not been a recent blood transfusion since a delayed haemolytic transfusion reaction can also cause spherocytosis. In the neonatal period, consider haemolytic disease of the newborn, particularly that due to ABO incompatibility. 

Hereditary elliptocytosis 

The term hereditary elliptocytosis covers a heterogeneous group of inherited red cell membrane abnormalities characterized by elliptical red cells (see Fig. 2.14). Inheritance is usually autosom- al dominant. Cases occur in many ethnic groups. Most cases are not anaemic and many do not have significant haemolysis. The blood film shows elliptocytes and some ovalocytes. Poly- chromasia is usually absent. Cases with haemolytic anaemia have polychromasia and, in addition to elliptocytes and ovalo- cytes, often have other poikilocytes including some red cell fragments. In the great majority of cases the FBC is normal. Anaemic cases show a reduced Hb and increased RDW. 

Further steps: The diagnosis of hereditary elliptocytosis can usually be made from the blood film. As haemolysis is unusual, further diagnostic tests are not usually necessary. 

Glucose-6-phosphate dehydrogenase deficiency 

Glucose-6-phosphate dehydrogenase (G6PD) is a red cell enzyme necessary to protect haemoglobin and other red cell proteins from endogenous or exogenous oxidant stress. Deficiency of G6PD leaves the individual susceptible to haemolysis during infection (endogenous oxidants generated by neutrophils) or on exposure to certain drugs, naphthalene in mothballs or fava beans (exogenous oxidants). Inheritance is sex-linked recessive. Cases occur in many ethnic groups but particularly in Africans, Afro-Caribbean, Afro-Americans, and in populations from the Middle East and around the Mediterranean. Most patients with a deficiency are haematologically normal between attacks of acute haemolysis. 

During a haemolytic crisis the blood film (Fig. 4.7) shows irregularly contracted cells, keratocytes (‘bite cells’) and red cells with the haemoglobin retracted into half of the red cell mem- brane (hemighosts). The irregularly contracted cells often have protrusions that indicate the presence of Heinz bodies attached to the red cell membrane. Heinz bodies are red cell inclusions composed of denatured haemoglobin. They can be identified by a specific supravital stain. Heinz bodies and severely damaged red cells are removed by the spleen so that after a few days there are fewer irregularly contracted cells but polychromasia, indicative of a reticulocyte response, is then apparent. 

Further steps: A test for Heinz bodies is not always necessary as it is often obvious from the very typical blood film that the patient has haemolysis induced by an oxidant. An assay for G6PD should be done. However, if the patient is already recover-

Fig. 4.7  Blood film during an acute haemolytic episode in glucose-6- phosphate dehydrogenase (G6PD)  deficiency showing anaemia, irregularly contracted cells  (some  with protrusions) and  a hemighost.

ing from the haemolysis and has a high reticulocyte count the result may be normal. This is particularly likely to occur with the type of G6PD deficiency that is found in those of African ethnic origin; they generally have normal levels of G6PD in reticulocytes although older cells are deficient; an episode of acute haemolysis can thus paradoxically lead to a rise in the G6PD concentration. Therefore, if the history and blood film suggest the possibility of G6PD deficiency but the assay is nor- mal, repeat the test when the patient has recovered from the episode of haemolysis and the reticulocyte count has gone back to normal. 

Autoimmune haemolytic anaemia 

Autoimmune haemolytic anaemia is anaemia caused by autoan- tibodies, active at body temperature, directed at red antigens (warm autoantibodies). The condition is uncommon and can occur at any age. 

The blood film (Fig. 4.8) shows spherocytosis and polychro- matic macrocytes. The blood film in autoimmune haemolytic

Fig. 4.8  Autoimmune haemolytic anaemia showing anaemia, spherocytosis and  several polychromatic macrocytes.

anaemia cannot be reliably distinguished from that of hereditary spherocytosis, although anaemia and spherocytosis are often more severe than is usual in hereditary spherocytosis. Occasion- ally there are small red cell agglutinates or red cells that have been ingested by monocytes. The blood count shows the same abnormalities as are present in hereditary spherocytosis. 

Further steps: Check the direct antiglobulin test (Coombs’ test) to see if there is immunoglobulin or complement on the surface of the red cells. Test for antinuclear activity and for the presence of antibodies to double-stranded DNA, as an autoim- mune haemolytic anaemia is often a feature of systemic lupus erythematosus. 

Beta thalassaemia major 

Beta thalassaemia major is a severe, transfusion-dependent anae- mia resulting from homozygosity or compound heterozygosity for beta thalassaemia (b0b0 or b0b+ or b+b+). It occurs in all the ethnic groups with a significant incidence of beta thalassaemia trait. Clinical features are those of severe anaemia and, in addi- tion, hepatomegaly, splenomegaly and overexpansion of mar- row-containing bones. 

The blood film shows striking anisocytosis, poikilocytosis, hypochromia and microcytosis. Poikilocytes include target cells. Red cells often contain Pappenheimer bodies and show ba- sophilic stippling. In patients who have also been splenect- omized, Pappenheimer bodies are very numerous and Howell–Jolly bodies are present. Sometimes there are precipi- tates with the same staining characteristics as haemoglobin; these are alpha chain precipitates, formed because of the lack of the beta globin chains that would normally combine with the alpha chains. NRBC are common. They show defective haemo- globinization and dysplastic features such as nuclear lobulation and fragmentation. There is marked reduction of the RBC, Hb, PCV/Hct, MCV and MCH. The MCHC is also reduced and the RDW is increased. 

In patients who are being transfused (Fig. 4.9) there will be a mixture of normal transfused cells and the patient’s cells show- ing the above abnormalities. 

Further steps: In the absence of transfusion, beta thalassaemia major is incompatible with life so the diagnosis is usually made in infancy. The affected infants are found to become anaemic from around 6 months of life, as synthesis of fetal haemoglobin stops and synthesis of adequate amounts of haemoglobin A does not occur. The infant requires haemoglobin electrophoresis or high performance liquid chromatography (HPLC). This will show either no haemoglobin A or only small amounts with the only haemoglobin present in significant amounts being haemo- globin F. Both parents should be tested for beta thalassaemia trait and genetic counselling should be given. Beyond the neonatal period, the differential diagnosis is with beta thalassaemia inter- media. This is a genetically heterogeneous group of disorders that are more severe than beta thalassaemia trait but are compat- ible with life, even if not transfused.

Fig. 4.9  Dimorphic blood  film in a patient with thalassaemia major who was  being  transfused. The  transfused red cells  appear normal whereas the patient’s red cells  show hypochromia, anisocytosis, poikilocytosis and Pappenheimer bodies. One  hypochromic cell  (bottom right)  contains an alpha chain precipitate. There are three NRBC,  which show cytoplasmic defects consequent on the  failure of haemoglobin synthesis.

Sickle cell anaemia 

Sickle cell anaemia results from homozygosity for an abnormal beta globin gene, the bS gene. As there is no normal beta gene there is no synthesis of normal beta globin and consequently no haemoglobin A can be produced. The haemoglobin is almost all haemoglobin S with a small amount of haemoglobin A2 and a variable, sometimes increased, amount of haemoglobin F. Sickle cell anaemia is commonest in those of African ancestry but occurs also in other ethnic groups including Indians, Arabs and Greeks. The most prominent clinical feature is recurrent painful crises caused by tissue infarction. The blood film in sickle cell anaemia (see Fig. 2.21) shows anaemia, sickle cells, boat-shaped cells and target cells. Once infancy is past, the changes of hyposplenism are also present, as a result of splenic infarction. Most striking is the presence of Howell–Jolly bodies but there are also some Pappenheimer bodies. NRBC are usually present and the WBC, neutrophil count, lymphocyte count and platelet count are often elevated. Polychro- masia is usually present. The lack of polychromasia in sickle cell anaemia is a significant finding since it may indicate that red cell production has ceased and severe anaemia is developing, usually as a consequence of intercurrent parvovirus B19 infection. 

The blood count usually shows an Hb of 7–9 g/dl. Some pa- tients have a reduced MCV and MCH. The RDW is increased. 

Further steps: Do haemoglobin electrophoresis or HPLC anal- ysis to demonstrate the absence of haemoglobin A and the pres- ence of mainly haemoglobin S with some haemoglobin A2 and F. Check parents and confirm that both carry haemoglobin S. If the MCV and MCH are normal or near normal the diagnosis of sickle cell anaemia is confirmed but if the patient has microcytic red cells the diagnosis could also be compound heterozygosity for haemoglobin S and beta0 thalassaemia. Family studies may per- mit the distinction but if this is not possible DNA analysis can be carried out. In an emergency, the diagnosis of sickle cell anaemia can be made fairly reliably by consideration of the Hb, red cell indices, blood film and sickle solubility test. 

Sickle cell trait 

Sickle cell trait is an asymptomatic condition consequent on heterozygosity for the bS gene. Haemoglobin S and haemoglobin A are present in similar amounts although there is somewhat more haemoglobin A than haemoglobin S. The blood film may either be normal or show microcytosis or target cells. The FBC may be normal or show microcytosis. Since both the blood film and the blood count may be normal it is clear that the diagnosis of sickle cell trait cannot be based on either but requires specific tests for its detection. 

Further steps: Haemoglobin electrophoresis or HPLC is re- quired. The nature of a variant haemoglobin with characteristics suggestive of haemoglobin S on one of these tests must be con- firmed by a sickle solubility test or by use of two independent methods, e.g. both haemoglobin electrophoresis and HPLC. Sickle cell trait (haemoglobin S less than 50%) has to be distin- guished from sickle cell/beta+ thalassaemia compound heterozy- gosity (haemoglobin S greater than 50%). 

In an emergency, sickle cell trait can be provisionally diag- nosed from the Hb, red cell indices, blood film and sickle solubil- ity test. Sickle cell/haemoglobin C compound heterozygosity can also have a normal Hb and red cell indices but the blood film is much more abnormal than that of sickle cell trait (see below). 

Sickle cell/haemoglobin C disease 

Patients who are compound heterozygotes for haemoglobin S and haemoglobin C have these two haemoglobins in approxi- mately equal amounts. Since they have no normal beta genes they cannot produce haemoglobin A. Sickle cell/haemoglobin C disease occurs in those with West African ancestry (since haemo- globin C originated in West Africa). Symptoms can be similar to those of sickle cell anaemia but are usually milder. 

The blood film shows target cells, irregularly contracted cells and boat-shaped cells but classic sickle cells are much less fre- quent than in sickle cell anaemia. Many patients have a variable number of typical poikilocytes containing both haemoglobin S and haemoglobin C (SC poikilocytes) (see Fig. 2.22). There may be NRBC, polychromasia and features of hyposplenism but these are all less prominent than in sickle cell anaemia. Rare red cells may be found containing haemoglobin C crystals, recog- nized by their parallel edges. The FBC may show a normal Hb or a mild anaemia, the Hb being generally higher than 8 g/dl. Some patients have a reduced MCV and some have an elevated MCHC. 

Further steps: Haemoglobin electrophoresis or HPLC, supple- mented by a sickle solubility test, is required. Two independent tests are required to confirm the diagnosis. 

Haemoglobin C disease 

Haemoglobin C disease is consequent on homozygosity for an abnormal beta gene, bC. There is no haemoglobin A. This condi-tion occurs in people of West African ancestry. There is a chronic haemolytic anaemia, which may be asymptomatic or lead to gallstones. 

The blood film (see Fig. 2.13) usually shows a mixture of target cells and irregularly contracted cells. Rare cells may contain haemoglobin C crystals. The FBC usually shows a mild to mod- erate anaemia. The MCV and MCH are often reduced and the MCHC is often increased. 

Further steps: Haemoglobin electrophoresis or HPLC is re- quired. Two independent tests are required to confirm the diag- nosis. If there is microcytosis, an alternative diagnosis of haemoglobin C/beta0 thalassaemia must be considered. 

Abnormalities of white cells 

Neutrophil leucocytosis (neutrophilia) 

An increased neutrophil count is usually caused by increased bone marrow output. However, it can also be caused both by mobilization of the marginated granulocyte pool (neutrophils which have been adherent to the endothelium), e.g. following vigorous exercise or an epileptic fit, and by decreasing egress of neutrophils to the tissues, e.g. after administration of high doses of corticosteroids. Some important causes of neutrophil leucocy- tosis are shown in Table 4.4. 

Bacterial infection 

Most patients with bacterial infection have a neutrophil leucocy- tosis. The blood film may also show a left shift, toxic granula- tion, Döhle bodies and vacuolation (Fig. 4.10). There may be small numbers of atypical lymphocytes (e.g. plasmacytoid lym- phocytes). There is lymphopenia and eosinopenia. If the bacterial infection becomes more chronic there may also be monocytosis, anaemia and increased rouleaux formation. It is important not to misinterpret the physiological changes of pregnancy and the post-partum period as being due to infection. It should also be noted that all the changes characteristic of

Table 4.4  Some  important causes of neutrophil leucocytosis.

Fig. 4.10  Blood film in acute infection showing neutrophil leucocytosis, toxic granulation and  vacuolation.

infection can be caused by the administration of granulocyte and granulocyte–macrophage colony-stimulating factors. 

Further steps: Consider the clinical setting to exclude causes of neutrophilia other than bacterial infection. Look for other blood film features typical of bacterial infection.

Chronic myeloid leukaemia 

Chronic myeloid leukaemia (also called chronic granulocytic leukaemia because the main cells produced are granulocytes) is a distinctive neoplastic condition, which, in the great majority of cases, is associated with a specific acquired cytogenetic abnor- mality in all myeloid cells. This is a translocation between chromosomes 9 and 22, known as t(9;22), leading to the forma- tion of an abbreviated chromosome 22 known as the Philadel- phia (Ph) chromosome. 

Chronic myeloid leukaemia occurs at any age, mainly from late adolescence to old age. The disease is rare in children but occasional cases do occur. Clinically there is marked splenome- galy and a lesser degree of hepatomegaly. The blood film is very characteristic (Fig. 4.11) and a correct diagnosis can usually be made from the blood count and film alone. There is a marked leucocytosis with the most frequent cells being myelocytes and mature neutrophils. There is a less marked increase in metamyelocytes, promyelocytes and blast cells. Basophils are increased in number in virtually all cases and eosinophils in about 80% of cases. Monocytes are increased,

Fig. 4.11  Chronic myeloid (chronic granulocytic) leukaemia showing three basophils, an eosinophil myelocyte and  mature and  immature cells  of neutrophil lineage.

but not in proportion to cells of the granulocyte lineages. There is usually a mild to moderate normocytic, normochromic anaemia. The platelet count is most often normal or increased but is occasionally reduced. The white cells do not usually show any dysplastic features, nor are there any reactive or ‘toxic’ features such as toxic granulation, Döhle bodies or neutrophil vacuolation. 

Further steps: A very accurate diagnosis is required because there is now very specific treatment for this type of leukaemia. All patients in whom the diagnosis is suspected require cytoge- netic analysis of bone marrow cells to detect the t(9;22) translo- cation. If it is not detected but the suspicion of the diagnosis is strong, molecular genetic analysis is also needed to see if the fusion gene that usually results from a t(9;22) transloca- tion (BCR–ABL fusion) is present despite the absence of the translocation. 

Chronic myeloid leukaemia is not usually confused with reac- tive neutrophilia, e.g. as a response to infection, because an increase in the eosinophil and basophil counts does not generally occur in infection, the number of granulocyte precursors in the blood is generally less and reactive changes such as toxic granu- lation, are often present. 

Lymphocytosis and morphologically abnormal lymphoid cells 

Lymphocytosis can be caused by increased mobilization of lym- phocytes from tissues into the blood stream or by increased production of lymphocytes, either in response to an antigenic stimulus or as a neoplastic condition. Transient lymphocytosis, due to redistribution of lymphocytes, occurs as an acute response to severe physical stress. When there is lymphocytosis as a re- sponse to an infection there are often also morphological changes in lymphocytes; these are most striking in infectious mononucl- eosis, in which atypical lymphocytes (see Fig. 3.9) are numerous. More subtle reactive changes in lymphocytes are common in other infections, particular infections in children or viral infec- tions at any age. Lymphoid cells are also morphologically abnor-

Table 4.5  Some  important causes of lymphocytosis.

mal in lymphoid neoplasms. Some of the important causes of lymphocytosis are shown in Table 4.5. 

Further steps: Assess the clinical history and physical findings. Has there been sudden severe physical stress? Are there signs or symptoms of infection, such as fever or sore throat? Is there enlargement of lymph nodes, liver or spleen, suggestive of a lymphoproliferative disorder? The blood count needs to be re- peated to exclude transient lymphocytosis. Persistent lymphocy- tosis is usually an indication for immunophenotyping the cells to facilitate diagnosis of a lymphoproliferative disorder. 

Chronic lymphocytic leukaemia 

Chronic lymphocytic leukaemia is a disease of the middle-aged and elderly. In the early stages of the disease there may be no abnormal physical findings. Later there is lymphadenopathy, hepatomegaly and splenomegaly. The blood film in chronic lymphocytic leukaemia (Fig. 4.12) is characterized by an increase of small, mature lymphocytes (which are of B lineage). In the early stages the lymphocyte count is only moderately elevated but later the count may be very high.

Fig. 4.12  Chronic lymphocytic leukaemia, showing lymphocytosis with an increase of mature small lymphocytes. There are two  smear cells.

The cells are more uniform in appearance than normal small lymphocytes. Both the cell and the nucleus have a smooth regu- lar outline. The nucleocytoplasmic ratio is high. The nuclear chromatin is usually moderately condensed and nucleoli are not usually apparent. Often nuclear chromatin is coarsely clumped, giving a mosaic or paving-stone pattern. The cytoplasm is agran- ular but occasionally contains crystals or globular inclusions. Smear cells, formed when the cell is disrupted during the spread- ing of the film, are characteristic but not pathognomonic. In some patients there is anaemia or thrombocytopenia. Anaemia can be caused by complicating autoimmune haemolytic anaemia and in these cases spherocytes are apparent. 

Further steps: If the lymphocytosis is persistent, immunophe- notyping is usually indicated to confirm the diagnosis, even if the patient does not have lymphadenopathy or hepatosplenome- galy. However, elderly patients with early disease need to be reassured as disease progression is often slow and they may not need treatment for many years, if at all. Nevertheless, confirm- ing the diagnosis means that the patient’s general practitioner is alert to the possibility of common complications, such as herpes zoster, that may need treatment. A bone marrow aspirate and trephine biopsy does not yield much information in the early stages of the disease, but these investigations are indicated if there is a possibility that the patient will soon need treatment. If there are spherocytes, a direct antiglobulin test is indicated to confirm the presence of complicating autoimmune haemolytic anaemia. 

Prolymphocytic leukaemia 

Prolymphocytic leukaemia is a disease of the middle-aged and elderly. Clinically there is marked splenomegaly and minor lym- phadenopathy. 

The WBC is usually markedly elevated and there is an increase of lymphoid cells, which are morphologically very abnormal (Fig. 4.13). They are larger than normal lymphocytes with a large and prominent nucleolus. The chromatin shows irregular condensation.

Fig. 4.13  Prolymphocytic leukaemia (B-lineage)  showing three prolymphocytes. These cells  are larger  than the  cells  of chronic lymphocytic leukaemia and  have  large,  prominent nucleoli.

Further steps: Immunophenotyping is indicated to confirm that there is a clonal B-cell population. 

Follicular lymphoma 

Lymphomas are lymphoid neoplasms that predominantly affect lymph nodes and other lymphoid tissues. However, lymphomas may infiltrate the bone marrow and there may be an overspill of lymphoma cells into the blood. Follicular lymphoma is a disease of adult life. It is characterized clinically by lymphadenopathy, splenomegaly or both, and pathologically by a nodular or follicu- lar growth pattern of neoplastic cells in lymph nodes. A signifi- cant minority of patients have circulating lymphoma cells. 

Follicular lymphoma cells are usually smaller than the cells of chronic lymphocytic leukaemia and more pleomorphic. They have very scanty cytoplasm. The nucleus shows a more even chromatin distribution. Some cells have distinctive notches or deep clefts (Fig. 4.14). Patients may have only small numbers of circulating lymphoma cells or the abnormal cells may be suffi- ciently numerous to cause a lymphocytosis. In those cases with lymphocytosis it is important to note the distinctive cellular features to avoid confusion with chronic lymphocytic leukaemia. 

Further steps: Immunophenotyping is indicated to confirm that there is a clonal B-cell population. Lymph node biopsy may

Fig. 4.14  Cleft lymphocyte in follicular lymphoma.

be needed to confirm the diagnosis. However, specific cytogenet- ic and molecular genetic abnormalities are found in follicular lymphoma and these analyses, together with immunophenotyp- ing, can confirm the diagnosis and obviate the need for a general anaesthetic for a lymph node biopsy. 

Hairy cell leukaemia 

Hairy cell leukaemia is a B-lineage lymphoid neoplasm with distinctive neoplastic cells. It is a disease of adult life character- ized clinically by splenomegaly without lymphadenopathy. 

Hairy cells (Fig. 4.15) are larger than normal lymphocytes. The nucleus is often round but is sometimes lobulated or shaped like a peanut shell or a dumbbell. There is moderately plentiful, weakly basophilic cytoplasm with irregular ‘hairy’ margins. Hairy cells are usually present only in small numbers so a careful search may be necessary to find and identify them. Pancytopenia is usual with neutropenia being common and monocytopenia being particularly severe. 

Further steps: Immunophenotyping is indicated to confirm that there is a clonal B-cell population with a distinctive immu- nophenotype. Cytochemistry, to demonstrate tartrate-resistant acid phosphatase activity, can also confirm the diagnosis when considered in conjunction with the cytological features. A tre- phine biopsy is also diagnostically useful, showing characteristi- cally widely spaced cells.

Fig. 4.15  Two  hairy cells  in hairy cell  leukaemia.

Multiple myeloma 

Multiple myeloma is a plasma cell neoplasm in which the malig- nant cells usually secrete an abnormal immunoglobulin known as a paraprotein. The main site of disease is the bone marrow and bones. Multiple myeloma is predominantly a disease of the mid- dle-aged and elderly. Common clinical features are anaemia, bone pain, pathological fractures, hypercalcaemia and renal failure. 

In the majority of patients the blood film shows increased rouleaux formation and increased background staining between the cells. Both are caused by the increased concentration of immunoglobulin in the blood. There is a normocytic, normo- chromic anaemia. Occasionally there are circulating myeloma cells (see Fig. 2.4), which resemble normal plasma cells to a greater or lesser extent, i.e. they may have plentiful basophilic cytoplasm, an eccentric nucleus and a paler-staining zone in the cytoplasm adjacent to the nucleus representing the Golgi zone. 

Detecting the features of multiple myeloma in patients with a normocytic normochromic anaemia can be very important in patient management, as this is often the first clue to the nature of the patient’s illness. 

Further steps: The suspicion of multiple myeloma requires: a bone marrow aspirate and trephine biopsy, investigations for a serum paraprotein and for urinary monoclonal immunoglobulin light chains (Bence–Jones protein), measurement of the concen- tration of normal serum immunoglobulin and radiographs of relevant bones, including the skull (a ‘skeletal survey’). Occa- sionally magnetic resonance imaging (MRI), to image the bone marrow, may be necessary.

The acute leukaemias and related conditions 

The acute leukaemias are characterized by proliferation of im- mature cells, either lymphoid or myeloid, with a failure of differ- entiation to mature end cells. Because the immature cells are proliferating in the bone marrow they replace normal haemopoi-etic cells and cause anaemia and various cytopenias. Prolifera- tion of leukaemic cells in other organs causes some degree of hepatomegaly and splenomegaly and, particularly in the case of acute lymphoblastic leukaemia, lymphadenopathy. 

Acute lymphoblastic leukaemia 

Acute lymphoblastic leukaemia is predominantly a disease of children. It is caused by proliferation, in the bone marrow and lymphoid tissues, of lymphoblasts of either B or T lineage. Over- spill into the blood occurs in the majority of cases. Patients are often anaemic or thrombocytopenic. Acute lymphoblastic leu- kaemia has been divided into three morphological subtypes L1, L2 and L3 by an international cooperative group, the French– American–British (FAB) group. Most childhood cases have L1 morphology (Fig. 4.16). The blasts are fairly uniform in appear- ance but vary in size from that of a normal lymphocyte to about twice this size. They have a high nucleocytoplasmic ratio, a de- licate diffuse chromatin pattern and sometimes small nucleoli.

Fig. 4.16  Acute lymphoblastic leukaemia of L1 subtype. There is one

NRBC;  all the  other cells  are lymphoblasts.

Fig. 4.17  Acute lymphoblastic leukaemia of L2 subtype

The smaller blasts can show some chromatin condensation. Some childhood cases and a larger proportion of adult cases have L2 morphology (Fig. 4.17). The blasts are larger than those of L1, have more plentiful cytoplasm and are more pleomorphic. Both cells and nuclei may be irregular in shape and nucleoli are some- times prominent. A small minority of cases have L3 morphology (Fig. 4.18). Cells are fairly regular in shape. They have moderate- ly to strongly basophilic cytoplasm and prominent cytoplasmic

Fig. 4.18  Acute lymphoblastic leukaemia of L3 subtype (can also  be regarded as the  leukaemic equivalent of Burkitt’s lymphoma).

vacuolation in at least a proportion of cells. Whether blast cells have L1 or L2 features is of little clinical significance. L1 acute lymphoblastic leukaemia can usually be readily diagnosed from the cytological features alone, whereas L2 acute lymphoblastic leukaemia is more likely to be confused with acute myeloid leukaemia and special tests to make the distinction are impor- tant. Otherwise, this categorization can be ignored. The L3 subtype, however, has a very specific clinical significance. Cyto- logical features are the same as in the leukaemic phase of Burkitt’s lymphoma. L3 morphology correlates with a mature B cell immunophenotype and requires specific management. In fact, the most recent classification of acute leukaemia, that pro- posed by the World Health Organization expert group, classifies this condition as a leukaemic phase of non-Hodgkin’s lymphoma rather than as acute leukaemia. This is appropriate since, al- though the disease is clinically very aggressive, the immunophe- notype is that of a mature B cell rather than that of a B-cell precursor. 

Further steps: When facilities are available, immunophenotyp- ing is always indicated in suspected acute lymphoblastic leukae- mia. This is for two reasons; (i) to distinguish the blast cells of T- or B-lineage acute lymphoblastic leukaemia from the blast cells of some acute myeloid leukaemias with very primitive myelob- lasts; and (ii) to distinguish non-Hodgkin’s lymphoma (mature B or T cells) from acute lymphoblastic leukaemia/lymphoblastic lymphoma (B- or T-cell precursors). 

These are very important distinctions to make since the treat- ment is very different. In addition, if facilities are available, cytogenetic and molecular analysis are indicated in all cases of acute lymphoblastic leukaemia. This is because all cases are not the same. There are many sub-types that require individual man- agement. Cytochemistry is irrelevant in acute lymphoblastic leukaemia unless immunophenotyping is unavailable. 

Acute myeloid leukaemia 

Acute myeloid leukaemia occurs at all ages from the neonatal period to old age. However, the incidence increases steadily

Table 4.6  Simplified FAB classification of acute myeloid leukaemia.

through adult life and old age. Acute myeloid leukaemia has been divided by the FAB group into eight morphological sub- types, which are summarized, in a simplified form, in Table 4.6. Diagnosis and classification of acute myeloid leukaemia require examination of a bone marrow aspirate but, since blast cells are usually present in the blood, a provisional diagnosis can often be made from examination of the blood film. It is necessary to recognize myeloblasts, monoblasts and normal and abnormal promyelocytes in order to recognize and classify acute myeloid leukaemia. In M0 and M1 acute myeloid leukaemia the predom- inant cell is a myeloblast. It is a large cell with a high nucleocy- toplasmic ratio. One or more nucleoli may be detected in the nucleus. In M1 acute myeloid leukaemia the cytoplasm may contain scanty granules or Auer rods (Fig. 4.19). In M2 acute myeloid leukaemia promyelocytes are also present (Fig. 4.20). They have more numerous granules than myeloblasts and may have an eccentric nucleus and a Golgi zone. In M4 acute myeloid leukaemia both myeloblasts and monoblasts are present (Fig. 4.21). Monoblasts are larger than myeloblasts with voluminous cytoplasm. Cytoplasmic basophilia varies from weak to moder- ately strong. The cytoplasm is sometimes vacuolated. The monoblast may be a round cell with a round nucleus or irregular in shape with a lobulated nucleus. There is often a large nucleo- lus. In M5 acute myeloid leukaemia the dominant cell may be a monoblast (M5a) or there may also be promonocytes and mature monocytes (M5b). Promonocytes are larger than monocytes and

Fig. 4.19  Acute myeloid leukaemia of M1 subtype showing six myeloblasts and  a lymphocyte. The  blast cell  adjacent to the  lymphocyte contains an Auer  rod.

Fig. 4.20  Acute myeloid leukaemia of M2 subtype showing two promyelocytes.

Fig. 4.21  Acute myeloid leukaemia of M4 subtype showing a myeloblast

(left) and  two  monoblasts (right).

have more basophilic and heavily granulated cytoplasm. M3 acute myeloid leukaemia (Fig. 4.22) is cytologically very distinctive. The promyelocyte cytoplasm is packed with large, brightly staining azurophilic granules. There may be giant granules or bundles of Auer rods. M3 variant acute myeloid leukaemia is more difficult to diagnose on cytological features, particularly from the peripheral blood film. By light microscopy most of the promyelocytes have no apparent granules but a minority have fine dust-like granules, a pink blush to the cytoplasm or bundles of Auer rods. Many of the promyelocytes have a distinctive bilobed nucleus (Fig. 4.23). Most cases of acute myeloid leukaemia have a normocytic, normochromic anaemia and thrombocytopenia. A small minority of cases have an increased platelet count. Neutropenia is also characteristic but some cases of M2 acute myeloid leukaemia have neutrophilia. A very small minority of cases have eosinophilia or basophilia. M0 and M7 acute myeloid leukaemia cannot be distinguished from acute lymphoblastic leukaemia by microscopy alone. Diagnosis of M6 acute myeloid leukaemia always requires bone marrow examination.

Fig. 4.22  Acute myeloid leukaemia of M3 subtype showing hypergranular promyelocytes, one  of which contains a giant granule

Fig. 4.23  Acute myeloid leukaemia of M3 variant subtype showing the characteristic bilobed hypogranular promyelocytes.

Further steps: If acute myeloid leukaemia is suspected, cytochemistry is needed to confirm the myeloid nature of the abnormal cells (if this is not already obvious). A bone marrow aspirate and cytogenetic analysis are also needed, since the cytogenetic sub-type is increasingly used in planning optimal treatment for each individual patient. Immunophenotyping is sometimes needed, to confirm a diagnosis of acute myeloid rather than acute lymphoblastic leukaemia. This is necessarily so in the FAB category of M0 acute myeloid leukaemia, in which the blast cells are very primitive and do not express myeloperoxidase, nonspecific esterase or other myeloid enzymes. They do, however, express antigens that are characteristic of myeloid cells. Immunophenotyping may also be necessary in cases of acute myeloid leukaemia in which the leukaemic blast cells are megakaryoblasts (M7 acute myeloid leukaemia). 

If M3 or M3 variant acute myeloid leukaemia is suspected, it is vital to confirm the diagnosis rapidly. This is because this condition is often complicated by disseminated intravascular coagulation and there is a need for urgent correction of the coagulation abnormality and specific anti-leukaemic treatment. An accurate as well as a speedy diagnosis of this subtype of acute leukaemia is particularly important since the specific treatment indicated differs from that in other types of acute myeloid leu- kaemia. 

The myelodysplastic syndromes 

The myelodysplastic syndromes are related to acute myeloid leukaemia. Both are neoplasms of myeloid cells with continued proliferation of myeloid precursors but defective production of mature end cells. In the myelodysplastic syndromes the dissociation between proliferation and maturation is not as severe as in acute myeloid leukaemia so that some end cells are produced. However, haemopoiesis is ineffective, leading to the paradox of frequent pancytopenia (anaemia, leucopenia and thrombocytope- nia) despite a cellular bone marrow. Blast cells may be increased and acute myeloid leukaemia may supervene in patients with one of the myelodysplastic syndromes. This group of closely related conditions occurs mainly in the elderly.

The blood film shows various cytopenias, most often anaemia, neutropenia and thrombocytopenia. Blood cells are often mor- phologically abnormal. Red cells may be macrocytic or, in those with defective incorporation of iron into haemoglobin, there may be a minor population of hypochromic microcytes and Pap- penheimer bodies (see Fig. 2.23). Neutrophils may be hypogran- ular (see Fig. 1.13) or show the acquired Pelger–Huët anomaly. Platelets may show abnormal variation in size or be hypogranu- lar or agranular. Blasts may be present and occasionally they contain Auer rods. Monocytes may be increased. The neutrophil count may be increased but neutropenia is much more common. The platelet count is increased in a minority of patients but is more often decreased. 

Further steps: Bone marrow aspiration is required, to assess the number of blast cells and exclude a diagnosis of acute myeloid leukaemia. This should be supplemented by cytogenetic analy- sis, which sometimes confirms an otherwise uncertain diagnosis and in other instances gives information of prognostic impor- tance. Cytochemical stains are indicated on the bone marrow aspirate, to detect any ring sideroblasts (Perls’ stain) or Auer rods (Sudan Black B or myeloperoxidase stain) that might be present. Since non-neoplastic conditions can also cause dysplastic changes in haemopoietic cells, it is sometimes necessary to exclude other conditions before making a presumptive diagnosis of mye- lodysplastic syndrome. Such conditions include alcohol and drug toxicity, vitamin B12 or folic acid deficiency, heavy metal expo- 

sure and human immunodeficiency virus (HIV) infection.

Idiopathic myelofibrosis 

Idiopathic myelofibrosis is a myeloproliferative disorder with onset usually in middle or old age.The fibrosis that affects the bone marrow is a reactive change that results from the proliferation of a clone of neoplastic haemopoietic cells. It is therefore no longer ‘idiopathic’ but the name remains convenient, to dis- tinguish this condition from secondary myelofibrosis. The blood film is important in suggesting the diagnosis. There is a normocytic, normochromic anaemia with marked anisocytosis and poikilocytosis. Teardrop poikilocytes are present. The blood film is leucoerythroblastic, i.e. NRBC and granulocyte precursors are present. In the early stages of the disease there may be neu- trophilia or thrombocytosis but usually there is pancytopenia. Platelets may show increased variation in size and granularity with some poorly granulated platelets and some giant forms. Occasionally circulating megakaryocytes or megakaryocyte bare nuclei are present. 

Further steps: The blood film in idiopathic myelofibrosis can- not be distinguished from that in myelofibrosis secondary to other myeloproliferative disorders (e.g. polycythaemia vera or essential thrombocythaemia). Knowledge of the previous history is essential to make the distinction. The differential diagnosis also includes secondary myelofibrosis due to bone marrow infil- tration in metastatic carcinoma. Assessment of clinical features is very important in making this distinction since splenomegaly is almost invariable in idiopathic myelofibrosis but is rare in metastatic carcinoma. A leucoerythroblastic blood film can also result from shock or acute hypoxia or reflect recovery from bone marrow suppression or recovery from haematinic deficiency (deficiency of iron, vitamin B12 or folic acid).