In health, the red blood cells vary relatively little in size and shape (Fig. 5.1). In well-spread, dried and stained films the great majority of cells have round, smooth contours and diameters within the comparatively narrow range of 6.0–8.5 μm. As a rough guide, normal red cell size appears to be about the same as that of the nucleus of a small lymphocyte on the dried film (Fig. 5.1). The red cells stain quite deeply with the eosin component of Romanowsky dyes, particularly at the periphery of the cell as a result of the cell’s normal biconcavity. A small but variable proportion of cells in well-made films (usually <10%) are definitely oval rather than round, and a very small percentage may be contracted and have an irregular contour or appear to have lost part of their substance as the result of fragmentation (schistocytes). According to Marsh, the percentage of ‘pyknocytes’ (irregularly contracted cells) and schistocytes in blood from healthy adults does not exceed 0.1% and the proportion is usually considerably less than this, whereas in normal, full-term infants the proportion is higher, 0.3–1.9%, and in premature infants it is still higher, up to 5.6%.¹

Normal and pathological red cells are subject to considerable distortion in the spreading of a film and, as already mentioned, it is imperative to scan films carefully to find an area where the red cells are least distorted before attempting to examine the cells in detail. Such an area can usually be found toward the tail of the film, although not actually at the tail. Rouleaux often form rapidly in blood after withdrawal from the body and may be conspicuous even in films made at a patient’s bedside. They are particularly noticeable in the thicker parts of a film that have dried more slowly. Ideally, red cells should be examined in an area in which there are no rouleaux and the red cells are touching but with little overlap. The film in the chosen area must not be so thin as to cause red cell distortion; if the tail of the film is examined, a false impression of spherocytosis may be gained. The varying appearances of different areas of the same blood film are illustrated in Figures 5.2–5.4. The area illustrated in Figure 5.2 would clearly be the best for looking at red cells critically.

Figure 5.2 Photomicrograph of a blood film. Ideal thickness for examination.

Figure 5.3 Photomicrograph of a blood film showing an area that is too thin for examination (same film as Fig. 5.2).

Figure 5.4 Photomicrograph of a blood film showing an area that is too thick for examination (same film as Fig. 5.2).

The advantages and disadvantages of examining red cells suspended in plasma have been referred to briefly in Chapter 4 (see p. 63). By this means, red cells can be seen in the absence of artefacts produced by drying, and abnormalities in size and shape can be better and more reliably appreciated than in films of blood dried on slides. However, the ease and rapidity with which dried films can be made, and their permanence, give them an overwhelming advantage in routine studies.

In disease, abnormality in the red cell picture stems from four main causes, which lead to characteristic cytological abnormalities (Table 5.1).

Table 5.1 Mechanisms of red cell abnormalities and resultant cytological features

Cause	Resultant abnormality
Attempts by the bone marrow to compensate for anaemia by increased erythropoiesis	Signs of less mature cells in the peripheral blood (polychromasia and erythroblastaemia)
Inadequate synthesis of haemoglobin	Reduced or unequal haemoglobin content and concentration (hypochromia, anisochromasia or dimorphism)
Abnormal erythropoiesis, which may be effective or ineffective	Increased variation in size (anisocytosis) and shape (poikilocytosis), basophilic stippling, sometimes dimorphism
Damage to, or changes affecting, the red cells after leaving the bone marrow, including the effects of reduced or absent splenic function	Spherocytosis, irregular contraction, elliptocytosis, ovalocytosis or fragmentation (schistocytosis); the presence of Pappenheimer bodies, Howell–Jolly bodies and variable numbers of certain specific poikilocytes (target cells, acanthocytes and spherocytes)

Abnormal erythropoiesis

Anisocytosis (αυισoζ, unequal) and Poikilocytosis (πoικιλoζ, varied)

Anisocytosis and poikilocytosis are non-specific features of almost any blood disorder. The terms imply more variation in size or shape than is normally present (Figs 5.5, 5.6). Anisocytosis may be a result of the presence of cells larger than normal (macrocytosis), cells smaller than normal (microcytosis) or both; frequently both macrocytes and microcytes are present (Fig. 5.5).

Figure 5.5 Photomicrograph of a blood film from a patient with a myelodysplastic/myeloproliferative neoplasm, unclassified. Shows moderate anisocytosis, anisochromasia and poikilocytosis. There is one neutrophil band form.

Figure 5.6 Photomicrograph of a blood film from a patient with compound heterozygosity for haemoglobin E and β° thalassaemia. Shows marked anisocytosis and poikilocytosis.

Poikilocytes are produced in many types of abnormal erythropoiesis, for example, megaloblastic anaemia (Fig. 5.7), iron deficiency anaemia, thalassaemia, myelofibrosis (both idiopathic and secondary) (Fig. 5.8), congenital dyserythropoietic anaemia (Fig. 5.9) and the myelodysplastic syndromes. Elliptocytes and ovalocytes are among the poikilocytes that may be present when there is dyserythropoiesis; they are often present in megaloblastic anaemia (macro-ovalocytes) and in iron deficiency anaemia (‘pencil cells’), but they may also be seen in myelodysplastic syndromes and in primary myelofibrosis (Fig. 5.8). The number of elliptocytes and teardrop poikilocytes has been observed to correlate with the severity of iron deficiency anaemia.² Poikilocytes are not only characteristic of disordered erythropoiesis but are also seen in various congenital haemolytic anaemias caused by membrane defects and in acquired conditions such as microangiopathic haemolytic anaemia and oxidant damage; in these disorders, the abnormality of shape results from damage to cells after formation and is described later in this chapter.

Figure 5.7 Photomicrograph of a blood film. Pernicious anaemia. Shows marked anisocytosis, moderate poikilocytosis (including oval macrocytes and teardrop cells) and a megaloblast.

Figure 5.8 Photomicrograph of a blood film. Idiopathic myelofibrosis. Many of the erythrocytes are elliptical or oval.

Figure 5.9 Photomicrograph of a blood film. Congenital dyserythropoietic anaemia type II. Shows marked anisocytosis, marked poikilocytosis, one unusually large macrocyte and one severely hypochromic cell.

Macrocytes

Classically found in megaloblastic anaemias (Fig. 5.10), but macrocytes are also present in some cases of aplastic anaemia, myelodysplastic syndromes and other dyserythropoietic states. In patients being treated with hydroxycarbamide (previously known as hydroxyurea) the red cells are often macrocytic. A common cause of macrocytosis is excess alcohol intake, and it occurs in alcoholic and other types of chronic liver disease. In these conditions, the red cells tend to be fairly uniform in size and shape and there may also be stomatocytes (Fig. 5.11). In the rare type III form of congenital dyserythropoietic anaemia, some of the macrocytes are exceptionally large. Another rare cause of macrocytosis is benign familial macrocytosis.³ Macrocytosis also occurs whenever there is an increased rate of erythropoiesis, because of the presence of reticulocytes. Their presence is suspected in routinely stained films because of the slight basophilia, giving rise to polychromasia (see p. 86) and is easily confirmed by special stains (e.g. New methylene blue, see p. 33). These polychromatic macrocytes should be distinguished from other macrocytes because the diagnostic significance is quite different.

Figure 5.10 Photomicrograph of a blood film. Megaloblastic anaemia. Shows macrocytes, oval macrocytes and a hypersegmented neutrophil.

Figure 5.11 Photomicrograph of a blood film. Liver disease. Shows macrocytosis and stomatocytosis.

Microcytes

The presence of microcytes usually results from a defect in haemoglobin formation. Microcytosis is characteristic of iron deficiency anaemia (Fig. 5.12), various types of thalassaemia (Fig. 5.13), and severe cases of anaemia of chronic disease. Causes that are rarer include congenital and acquired sideroblastic anaemias. Microcytosis related to a defect in haemoglobin synthesis should be distinguished from red cell fragmentation or schistocytosis (see p. 80). Both abnormalities can lead to a reduction of the mean cell volume (MCV). However, it should be noted that a low MCV is common in association with a defect in haemoglobin synthesis, whereas it is uncommon in fragmentation syndromes because the fragments usually comprise only a small percentage of erythrocytes.

Figure 5.12 Photomicrograph of a blood film. Iron deficiency anaemia. Shows hypochromia, microcytosis and poikilocytosis.

Figure 5.13 Photomicrograph of a blood film. Compound heterozygosity for Hb D-Punjab/β thalassaemia. Shows anisocytosis, poikilocytosis, hypochromia, microcytosis and basophilic stippling.

Basophilic Stippling

Basophilic stippling or punctate basophilia means the presence of numerous basophilic granules distributed throughout the cell (Fig. 5.14); in contrast to Pappenheimer bodies (see below), they do not give a positive Perls’ reaction for ionized iron. Punctate basophilia has quite a different significance from diffuse cytoplasmic basophilia. It is indicative of disturbed rather than increased erythropoiesis. It occurs in many blood diseases: thalassaemia, megaloblastic anaemias, infections, liver disease, poisoning by lead and other heavy metals, unstable haemoglobins and pyrimidine-5′-nucleotidase deficiency.⁴

Figure 5.14 Photomicrographs of blood films. (A) β thalassaemia trait shows hypochromia, microcytosis and basophilic stippling. (B) Erythropoietic porphyria. Shows prominent basophilic stippling.

Inadequate haemoglobin formation

Hypochromia (Hypochromasia) (υπoρ, under)

The term hypochromasia, or now, more often, hypochromia, refers to the presence of red cells that stain unusually palely. (In doubtful cases, it is wise to compare the staining of the suspect film with that of a normal film stained at the same time.) There are two possible causes: a lowered haemoglobin concentration and abnormal thinness of the red cells. A lowered haemoglobin concentration results from impaired haemoglobin synthesis. This may stem from failure of haem synthesis – iron deficiency is a very common cause (Fig. 5.15) and sideroblastic anaemia (Fig. 5.16) is a rare cause – or failure of globin synthesis as in the thalassaemias (Fig. 5.17). Haemoglobin synthesis may also be impaired in chronic infections and other inflammatory conditions. Abnormally thin red cells (leptocytes) (see p. 82) can be the result of a defect in haemoglobin synthesis (e.g. in thalassaemias and iron deficiency) but they also occur in liver disease. It cannot be too strongly stressed that a hypochromic blood picture does not necessarily mean iron deficiency, although this is the most common cause. In iron deficiency, the red cells are characteristically hypochromic and microcytic, but the extent of these abnormalities depends on the severity; hypochromia may be minor and may be overlooked if the haemoglobin concentration (Hb) exceeds 100 g/l. In heterozygous or homozygous α+ thalassaemia, heterozygous α° thalassaemia or heterozygous β thalassaemia, hypochromia is often less marked, in relation to the degree of microcytosis, than in iron deficiency. The presence of target cells or basophilic stippling also favours a diagnosis of thalassaemia trait rather than iron deficiency. In homozygous β thalassaemia, the abnormalities are greater than in iron deficiency at the same Hb and nucleated red cells are usually present, whereas they are not a feature of iron deficiency. If the patient is being transfused regularly, normal donor cells will also be present, producing a dimorphic blood film (Fig. 5.18).

Figure 5.15 Photomicrograph of a blood film. Iron deficiency anaemia. Shows a marked degree of hypochromia, microcytosis, marked anisocytosis and mild poikilocytosis; there are some normally haemoglobinized cells.

Figure 5.16 Photomicrograph of a blood film. Acquired sideroblastic anaemia (refractory anaemia with ring sideroblasts). Shows a dimorphic blood film with a mixture of normochromic normocytic cells and hypochromic microcytes; there are also several polychromatic macrocytes.

Figure 5.17 Photomicrograph of a blood film. Haemoglobin H disease. Shows microcytosis, moderate hypochromia, moderate anisocytosis and some poikilocytes (including teardrop poikilocytes and red cell fragments).

Figure 5.18 Photomicrograph of a blood film. β thalassaemia major. Shows a dimorphic blood film. The normal cells are transfused cells. The patient’s own cells show severe hypochromia. There are two nucleated red blood cells.

Anisochromasia (αυισoζ, unequal) and Dimorphic Red Cell Population

A distinction should be made between anisochromasia, in which there is abnormal variability in staining of red cells, and a dimorphic picture, in which there are two distinct populations. Anisochromasia, in which some but not all of the red cells stain palely, is characteristic of a changing situation. It can occur during the development or resolution of iron deficiency anaemia (Fig. 5.19) or the anaemia of chronic disease. In thalassaemia trait, in contrast, anisochromasia is much less common. A dimorphic blood film can be seen in several circumstances. It can occur when an iron deficiency anaemia responds to iron therapy, after the transfusion of normal blood to a patient with a hypochromic anaemia (Fig. 5.19), and in sideroblastic anaemia (Fig. 5.20). In acquired sideroblastic anaemia as a feature of a myelodysplastic syndrome, the two populations of cells are usually hypochromic microcytic and normochromic macrocytic, respectively.

Figure 5.19 Photomicrograph of a blood film. Iron deficiency anaemia. Shows a constant gradation of haemoglobinization of cells (i.e. anisochromasia). There is one elliptocyte.

Figure 5.20 Photomicrograph of a blood film. Acquired sideroblastic anaemia. Shows two distinct populations of cells: hypochromic cells, which also tend to be microcytic, and normocytic normochromic cells.

Damage to red cells after formation

Poikilocytosis can result not only from abnormal erythropoiesis but also from damage to red cells after their formation. The damage may be consequent on an intrinsic abnormality of the red cell such as a haemoglobinopathy, a membrane defect or an enzyme defect that renders the cell prone to shape alteration. Poikilocytosis can also result from extrinsic causes, as when a red cell is damaged by drugs, chemicals or toxins; by heat; or by abnormal mechanical forces. Poikilocytes of specific shapes suggest different aetiological factors.

Hyperchromasia (Hyperchromia) (υπρ, over)

Unusually deep staining of the red cells with a lack of central pallor may be seen in two circumstances: first, in the presence of macrocytes and second, when cells are abnormally rounded. In macrocytosis, as in neonatal blood and megaloblastic anaemias, it is the increased red cell thickness that causes the hyperchromia, and the mean cell haemoglobin concentration is normal. When hyperchromia results from cells being of abnormal shape, the red cell thickness is greater than normal and the MCHC is increased. Abnormally rounded cells may be either spherocytes or irregularly contracted cells. The distinction between these two cell types is of diagnostic importance.

Spherocytosis (σϕαιρα, a sphere)

Spherocytes are cells that are more spheroidal (i.e. less disc-like) than normal red cells but maintain a regular outline. Their diameter is less and their thickness is greater than normal. Only in extreme instances are they almost spherical in shape. It is useful to draw a distinction between spherocytes of normal size and microspherocytes; the latter result from red cell fragmentation or from removal of a considerable proportion of the red cell membrane by splenic or other macrophages. Spherocytes may result from genetic defects of the red cell membrane as in hereditary spherocytosis (Fig. 5.21); from the interaction between immunoglobulin- or complement-coated red cells and phagocytic cells, as in delayed transfusion reactions, ABO haemolytic disease of the newborn (Fig. 5.22) and autoimmune haemolytic anaemia (Fig. 5.23); and from the action of bacterial toxins (e.g. Clostridium perfringens lecithinase; Fig. 5.24).

Figure 5.21 Photomicrograph of a blood film. Hereditary spherocytosis. Shows a moderate degree of spherocytosis and anisocytosis. Note the round contour of the spherocytes.

Figure 5.22 Photomicrograph of a blood film. ABO haemolytic disease of the newborn. Spherocytosis is intense, and there are several polychromatic macrocytes.

Figure 5.23 Photomicrograph of a blood film. Autoimmune haemolytic anaemia. Shows marked spherocytosis and anisocytosis. There are numerous polychromatic macrocytes.

Figure 5.24 Photomicrograph of a blood film. Clostridium perfringens septicaemia. Shows an extreme degree of spherocytosis; note the round contour of the spherocytes. A markedly dimorphic picture.

Spherocytes usually appear perfectly round in contour in stained films; they have to be carefully distinguished from both irregularly contracted cells and ‘crenated spheres’ or sphero-echinocytes (Fig. 5.25), which are the end result of crenation (see p. 82). Sphero-echinocytes develop as artefacts, especially in blood that has been allowed to stand before films are spread (Fig. 5.26). The blood film of a patient who has been transfused with stored blood may show a proportion of sphero-echinocytes (Fig. 5.27).

Figure 5.25 Photomicrograph of a blood film. Sodium chlorate poisoning. Shows sphero-echinocytes, one keratocyte and one nucleated red cell.

Figure 5.26 Photomicrograph of a blood film. Normal blood after 24 h at 20°C. Shows a marked degree of crenation; also degenerative changes in white cells.

Figure 5.27 Photomicrograph of a blood film. Post-transfusion, showing one sphero-echinocyte.

Irregularly Contracted Red Cells

There are a number of causes of irregularly contracted cells. In drug- or chemical-induced haemolytic anaemias, a proportion of the red cells are smaller than normal and unusually densely stained (i.e. they appear contracted) and their margins are slightly or moderately irregular and may be partly concave (Fig. 5.28). These may be cells from which Heinz bodies have been extracted by the spleen. Similar cells may be seen in films of some unstable haemoglobinopathies before splenectomy (e.g. that caused by the presence of Hb Köln or Hb St Mary’s; Fig. 5.29) and in haemoglobin E homozygosity (Fig. 5.30) and, to a lesser extent, haemoglobin E heterozygosity. Heinz bodies are not normally visible in Romanowsky-stained blood films, but they may be seen in such films as pale pink–staining bodies at the cell margin or even protruding from the erythrocytes in severe unstable haemoglobin haemolytic anaemias after splenectomy and in acute oxidant-induced haemolytic anaemia, including that occurring in deficiency of glucose-6-phosphate dehydrogenase. An extreme degree of irregular contraction is characteristic of severe favism or any other very acute haemolytic episode in individuals who are glucose-6-phosphate dehydrogenase deficient. It is typical to see cells in which the haemoglobin appears to have contracted away from the cell membrane, an appearance sometimes referred to as a hemi-ghost (Fig. 5.31); there may also be ghost cells – cells with almost empty membranes containing negligible haemoglobin. Irregularly contracted cells can be seen in small numbers in β thalassaemia trait and in heterozygosity for haemoglobin C. There may be a considerable number in haemoglobin C homozygosity and in this condition haemoglobin C crystals may also be seen (Fig. 5.32).

Figure 5.28 Photomicrograph of a blood film. Haemolytic anaemia caused by an overdose of phenacetin. Shows four irregularly contracted cells and crenation.

Figure 5.29 Photomicrograph of a blood film. An unstable haemoglobin haemolytic anaemia (haemoglobin St Mary’s). Shows several irregularly contracted cells.

Figure 5.30 Photomicrograph of a blood film. Haemoglobin E homozygosity showing four irregularly contracted cells and target cells.

Figure 5.31 Photomicrograph of a blood film. Acute drug-induced haemolysis in a patient with glucose-6-phosphate dehydrogenase deficiency. There are irregularly contracted cells, ghost cells (black arrows) and hemi-ghosts. Heinz bodies can be seen (blue arrows).

Figure 5.32 Photomicrograph of a blood film. Haemoglobin C disease (homozygosity for haemoglobin C). Shows many target cells, irregularly contracted cells and a crystal of haemoglobin C. Sometimes it is apparent that haemoglobin C crystals are within otherwise empty red cell membranes.

A type of irregular contraction of unknown origin has been described by the term ‘pyknocytosis’.⁵ The pyknocytes closely resemble chemically damaged red cells. As already mentioned (see p. 70), a small number of pyknocytes may be found in the blood of infants in the first few weeks of life, especially in premature infants. The term ‘infantile pyknocytosis’ refers to a transient haemolytic anaemia, related to glutathione peroxidase and selenium deficiency, affecting infants in whom many pyknocytes are present (Fig. 5.33).^5,⁶

Figure 5.33 Photomicrograph of a blood film. Infantile pyknocytosis. Shows irregularly contracted cells similar to those seen in chemical- or drug-induced haemolytic anaemias.

Elliptocytosis and Ovalocytosis

Elliptocytes are often present in large numbers in hereditary elliptocytosis (Fig. 5.34). In hereditary pyropoikilocytosis, elliptocytes are only one of the many types of poikilocyte present (Fig. 5.35). South-east Asian ovalocytosis is characterized by the presence of a variable number of elliptocytes, macro-ovalocytes and stomatocytes (Fig. 5.36). In all these conditions, the reticulocytes are round in contour (i.e. the cell assumes an abnormal shape only in the late stages of maturation). Although the causative condition is inherited, the abnormalities of red cell shape only become apparent as the cells reach maturity.

Figure 5.34 Photomicrograph of a blood film. Hereditary elliptocytosis. Many of the cells are elliptical, and others are oval.

Figure 5.35 Photomicrograph of blood film from a child with hereditary pyropoikilocytosis. Shows spherocytes, elliptocytes, red cell fragments and polychromatic macrocytes.

Figure 5.36 Photomicrograph of a blood film. South-east Asian ovalocytosis. Shows stomatocytes, macrostomatocytes and macro-ovalocytes.

Spiculated cells and red cell fragmentation

The terminology applied to spiculated cells has been confusing because the same terms have been used to designate different types of cells. For this reason the term ‘burr cell’ should be discarded and the terms recommended by Bessis ⁷ should be adopted. On the basis of scanning electron microscopy (discussed later), he distinguished four types of spiculated cell – schistocyte, keratocyte, acanthocyte and echinocyte. The term echinocyte is used for the crenated cell. It is differentiated from the acanthocyte on the basis of the number, shape and disposition of the spicules.

Schistocytosis (Fragmentation) (σχιστoζ, cleft)

Schistocytes or erythrocyte fragments are found in many blood diseases. They are smaller than normal red cells and of varying shape. Sometimes they have sharp angles or spines (spurs), and sometimes they are round in contour, usually staining deeply but occasionally palely as the result of loss of haemoglobin at the time of fragmentation. If they are both round and densely staining, they may be referred to as microspherocytes. They occur in the following situations:

1. In certain genetically determined disorders (e.g. thalassaemias, congenital dyserythropoietic anaemia and hereditary pyropoikilocytosis)

2. In acquired disorders of red cell formation when erythropoiesis is megaloblastic or dyserythropoietic

3. As the consequence of mechanical stresses (e.g. in the microangiopathic haemolytic anaemias; Figs 5.37–5.39) and in cardiac haemolytic anaemias that are usually caused by a perivalvular leak accompanied by turbulence of left ventricular flow (Fig. 5.40)

4. As the result of direct thermal injury, as in severe burns (Fig. 5.41).

Figure 5.37 Photomicrograph of a blood film. Microangiopathic haemolytic anaemia. Shows angular red cell fragments.

Figure 5.38 Photomicrograph of a blood film. Microangiopathic haemolytic anaemia in systemic lupus erythematosus. Shows one very dense microspherocyte and other red cell fragments.

Figure 5.39 Photomicrograph of a blood film. Microangiopathic haemolytic anaemia. Shows numerous bizarrely shaped red cell fragments.

Figure 5.40 Photomicrograph of a blood film. Postcardiac surgery haemolytic anaemia. Shows numerous irregularly shaped cell fragments.

Figure 5.41 Photomicrograph of a blood film. Severe burns. Shows many very small, rounded red cell fragments (microspherocytes) and one crenated cell.

In burns, schistocytes are often rounded, being either microspherocytes or very small disc-shaped fragments. In addition, erythrocytes may be seen to be budding off small rounded blebs of cytoplasm. Not infrequently, as, for instance in the haemolytic-uraemic syndrome in children, the blood picture is made more bizarre by the superimposition of varying degrees of echinocytic change. Schistocytes are also a feature of thrombotic thrombocytopenia purpura.⁸

Keratocytes (κραζ, horn)

Keratocytes have pairs of spicules, usually either one pair or two pairs. They may be formed either by removal of a Heinz body by the pitting action of the spleen (Fig. 5.42) or by mechanical damage (Figs 5.43, 5.44). The terms ‘helmet cell’ and ‘bite cell’ have sometimes been used to describe keratocytes.

Figure 5.42 Photomicrograph of a blood film. Keratocytes and irregularly contracted cells in a patient with haemolysis caused by glucose-6-phosphate dehydrogenase deficiency.

Figure 5.43 Photomicrograph of a blood film. Haemolytic anaemia caused by dapsone. Shows many irregularly contracted cells, three cells with haemoglobin retracted from the red cell membrane and one keratocyte.

Figure 5.44 Photomicrograph of a blood film. Two keratocytes in a patient with microangiopathic haemolytic anaemia.

Acanthocytosis (ακανθα, spine)

The term acanthocytosis was introduced to describe an abnormality of the red cell in which there are a small number of spicules of inconstant length, thickness and shape, irregularly disposed over the surface of the cell (Fig. 5.45). They are often associated with abnormal phospholipid metabolism⁹^–¹¹ or with inherited abnormalities of red cell membrane proteins, as in the McLeod phenotype, caused by lack of the Kell precursor (Kx).¹² They are present in varying numbers following splenectomy and in hyposplenism. A similar cell occurs in severe liver disease (‘spur cell’ anaemia).¹³

Figure 5.45 Photomicrograph of a blood film. Liver failure. Acanthocytes are conspicuous.

Echinocytosis (χινoζ, sea-urchin or hedgehog)

Echinocytosis or crenation describes the process by which red cells develop numerous short, regular projections from their surface (Figs 5.25, 5.26). First described by Ponder¹⁴ as disc-sphere transformation, crenation has many causes. A few crenated cells may be seen in many blood films, even in those from healthy subjects. Crenation regularly develops if blood is allowed to stand overnight at 20°C before films are made (Fig. 5.26). It may be a marked feature, for obscure and probably diverse reasons, in freshly made blood films of patients suffering from a variety of illnesses, especially uraemia. Marked echinocytosis has been reported in premature infants after exchange transfusion or transfusion of normal red cells.¹⁵ When crenation is superimposed on an underlying abnormality, the red cells may appear bizarre in the extreme.

Crenation also occurs as an artefact if red cells are washed free from plasma and suspended in 9 g/l NaCl between glass surfaces, particularly at a raised pH; it also occurs in the presence of traces of fatty substances on the slides on which films are made and in the presence of traces of chemicals that at higher concentrations cause lysis.

The end stages of crenation are the ‘finely crenated sphere’ and the ‘spherical form’, which closely resemble spherocytes. The disc-sphere transformation may be reversible (e.g. that produced by washing cells free from plasma), and in this respect the contracted ‘spherical form’ (which has not lost surface) is quite distinct from the ‘spherocyte’ (which has lost surface), although they may closely resemble one another in stained films.

If echinocytosis is observed in a film, it usually represents a storage artefact caused by delay in making the film. It is a warning that morphologic features in the blood film cannot be assessed reliably. If present in films made from fresh blood, it is a clinically significant observation.

Miscellaneous erythrocyte abnormalities

Leptocytosis (λπτoζ, thin)

The term leptocytosis has been used to describe unusually thin red cells, as in severe iron deficiency or thalassaemia in which the cells may stain as rings of membrane with a little attached haemoglobin with large, almost unstained, central areas (Fig. 5.46). They can also occur when there is an excess of membrane in relation to cytoplasm, as can occur in liver disease.

Figure 5.46 Photomicrograph of a blood film. β thalassaemia major, after splenectomy. Shows one target cell and cells grossly deficient in haemoglobin, which are leptocytes. There are some transfused cells.

Target Cells

The term target cell refers to a cell in which there is a central round stained area and a peripheral rim of haemoglobinized cytoplasm separated by non-staining or more lightly staining cytoplasm. Target cells result from cells having a surface that is disproportionately large compared with their volume. They may be normal in size, microcytic or macrocytic. They are seen in films in chronic liver diseases in which the cell membrane may be loaded with cholesterol (Fig. 5.47), in hereditary hypo-betalipoproteinaemia,¹⁶ and in varying numbers in iron deficiency anaemia and in thalassaemia (Fig. 5.46). They are often conspicuous in certain haemoglobinopathies (e.g. haemoglobin C/β° thalassaemia; Fig. 5.48), haemoglobin C disease (Fig. 5.49), haemoglobin H disease (Fig. 5.50), sickle cell anaemia, sickle cell/haemoglobin C disease (Fig. 5.51), sickle cell/β thalassaemia, and haemoglobin E disease. Smaller numbers are usual in haemoglobin C trait, haemoglobin E trait and postsplenectomy. Splenectomy in thalassaemia may result in an extreme degree of leptocytosis and target cell formation.

Figure 5.47 Photomicrograph of a blood film. Alcoholic liver disease. Shows many target cells.

Figure 5.48 Photomicrograph of a blood film. Haemoglobin C/β° thalassaemia compound heterozygosity showing target cells, irregularly contracted cells and one spherocyte.

Figure 5.49 Photomicrograph of a blood film. Haemoglobin C homozygosity. Shows numerous target cells and irregularly contracted cells.

Figure 5.50 Photomicrograph of a blood film. Haemoglobin H disease. Shows target cells, hypochromic cells and poikilocytes.

Figure 5.51 Photomicrograph of a blood film. Sickle cell/haemoglobin C disease. Shows a sickle cell, target cells and two SC poikilocytes.

Stomatocytosis (στoμα, mouth)

Stomatocytes are red cells in which the central biconcave area appears slit-like in dried films. In ‘wet’ preparations, the stomatocyte is a cup-shaped red cell. The slit-like appearance of the cell’s concavity, as seen in dried films, is thus to some extent an artefact. The term was first used to describe the appearance of some of the cells in a rare type of haemolytic anaemia, hereditary stomatocytosis.¹⁷ They are also a feature of south-east Asian ovalocytosis. They have been described as being particularly frequent in films of Australians of Mediterranean origin.^18,¹⁹ Subsequently, stomatocytes were recognized in acquired conditions and occasionally they are prominent (Fig. 5.52). They are observed in liver disease, in alcoholism,²⁰ and occasionally in the myelodysplastic syndromes. There is a suspicion that in some films the occurrence of stomatocytosis is an in vitro artefact because it is known that the change can be produced by decreased pH and as the result of exposure to cationic detergent-like compounds and non-penetrating anions.²¹

Figure 5.52 Photomicrograph of a blood film. Patient with chronic myelogenous leukaemia taking hydroxycarbamide. Shows stomatocytosis. Many of the cells have a slit-like central unstained area.

Sickle Cells

The varied film appearances in sickle cell anaemia are illustrated in Figures 5.53–5.55. Sickle cells are almost always present in films of freshly withdrawn blood of adults with homozygosity for haemoglobin S. However, sickle cells are usually absent in neonates and are rare in adult patients with a high haemoglobin F percentage. Sometimes many irreversibly sickled cells are present, and in all cases massive sickling takes place when the blood is subjected to anoxia (see p. 315). In films of fresh blood, the sickled cells vary in shape between boat-shaped forms and sickles. Target cells are also often a feature of blood films from patients with sickle cell anaemia, and Howell–Jolly bodies are found when there is splenic atrophy.

Figure 5.53 Photomicrograph of a blood film. Sickle cell anaemia (homozygosity for haemoglobin S). Shows a sickled cell, boat-shaped cells and a nucleated red cell and target cells.

Figure 5.54 Photomicrograph of a blood film. Sickle cell anaemia (homozygosity for haemoglobin S). Shows boat-shaped cells and a nucleated red blood cell.

Figure 5.55 Photomicrograph of a blood film. Sickle cell anaemia (homozygosity for haemoglobin S). Shows elliptical sickle cells, target cells and Pappenheimer bodies.

Haemoglobin C Crystals and SC Poikilocytes

In patients with homozygosity for haemoglobin C, target cells and irregularly contracted cells are usually numerous and there may be occasional straight-edged haemoglobin C crystals, either apparently extracellularly (Fig. 5.32) or within the ghost of a red cell. In patients who are compound heterozygotes for both haemoglobin S and haemoglobin C, the film sometimes resembles that of haemoglobin C disease (Fig. 5.32). In other patients, there are elliptical cells, rare sickle cells and sometimes distinctive SC poikilocytes (Fig. 5.56).

Figure 5.56 Photomicrograph of a blood film. Sickle cell/haemoglobin C disease showing SC poikilocytes and target cells.

Erythrocyte Inclusions

The possibility of sometimes suspecting the presence of Heinz bodies on a routinely stained film and the detection of haemoglobin crystals within red cells has already been mentioned. Alpha chain inclusions can also sometimes be seen, e.g. in β thalassaemia major following splenectomy. Other red cell inclusions include Howell–Jolly bodies and Pappenheimer bodies.

Howell–Jolly Bodies

Howell–Jolly bodies are nuclear remnants. They are small, round cytoplasmic inclusions that stain purple on a Romanowsky stain. They are regularly present after splenectomy and when there is splenic atrophy (Fig. 5.57). They may be seen in a small percentage of red cells in pernicious anaemia. Usually only a few such inclusions are present, but they may be numerous in cases of coeliac disease and in other conditions in which there is splenic atrophy and megaloblastosis.

Figure 5.57 Photomicrograph of a blood film. Postsplenectomy. Shows acanthocytes, a target cell and a Howell–Jolly body.

Pappenheimer Bodies

Pappenheimer bodies are small peripherally sited basophilic (almost black) erythrocyte inclusions. They are smaller than Howell–Jolly bodies. Usually only a small number are present in a cell. They are composed of haemosiderin and their presence is related to sideroblastic erythropoiesis and hyposplenism (Figs 5.58, 5.59). Sometimes they are found in the majority of circulating red cells. Their nature can be confirmed by means of a Perls’ stain. They correspond to the siderotic granules of siderocytes and are never distributed in large numbers throughout the cells as in classical punctate basophilia. However, a single cell may show both punctate basophilia and Pappenheimer bodies. With Perls’ stain, the former granules are pink, whereas the latter are blue.

Figure 5.58 Photomicrograph of a blood film. β thalassaemia major, patient of regular blood transfusions, showing Pappenheimer bodies in several poorly haemoglobinized cells.

Figure 5.59 Photomicrograph of a blood film. Autoimmune thrombocytopenic purpura, postsplenectomy, showing a Pappenheimer body and a Howell–Jolly body.

Rouleaux and Autoagglutination

Rouleaux occur to some extent in all films but increased rouleaux formation is significant. The differences between rouleaux and autoagglutination are described on p. 63, and there is usually no difficulty in determining which is which in stained films (Figs 5.60, 5.61). However, in myelomatosis and in other conditions in which there is intense rouleaux formation, the rouleaux may simulate autoagglutination. Even so, if the film, apparently showing autoagglutination, is carefully scanned, an area in which rouleaux can be clearly seen will almost certainly be found, emphasizing the importance of careful selection of the area of film to be examined.

Figure 5.60 Photomicrograph of a blood film. Increased rouleaux formation in a patient with bacterial infection.

Figure 5.61 Photomicrograph of a blood film. Shows massive autoagglutination in a patient with chronic cold haemagglutinin disease (compare with Fig. 5.60).

Changes associated with a compensatory increase in erythropoiesis

Polychromasia (πoλθζ, many)

The term polychromasia suggests that the red cells are being stained many colours. In practice, it means that some of the red cells stain shades of bluish grey (Fig. 5.62) – these are the reticulocytes. Cells staining shades of blue, ‘blue polychromasia’, are unusually young reticulocytes. ‘Blue polychromasia’ is most often seen when there is either an intense erythropoietic drive or when there is extramedullary erythropoiesis, as, for instance, in myelofibrosis or carcinomatosis. It should be noted that in certain circumstances the absence of polychromasia is significant; in a patient with severe anaemia it indicates that the bone marrow response is inadequate, e.g. in aplastic anaemia and pure red cell aplasia.

Figure 5.62 Photomicrograph of a blood film. Polychromasia. Some red cells stain shades of bluish grey.

Erythroblastaemia

Erythroblasts may be found in the blood films of almost any patient with a severe anaemia; they are, however, very unusual in aplastic anaemia, and their presence should lead to this diagnosis being doubted. They are more common in children than in adults, and large numbers are a very characteristic finding in haemolytic disease of the newborn. Small numbers can be found in the cord blood of normal infants, whereas quite large numbers are found in that of premature infants.

When large numbers of erythroblasts are present, many of them may be derived from extramedullary foci of erythropoiesis (e.g. in the liver and spleen). This is likely, for instance, in haemolytic disease of the newborn and primary myelofibrosis. In myelofibrosis and carcinomatosis, the number of erythroblasts is often disproportionately high for the degree of anaemia, and a few immature granulocytes are usually also present (designated leucoerythroblastic anaemia) (Fig. 5.63).

Figure 5.63 Photomicrograph of a blood film. Primary myelofibrosis. Shows a leucoerythroblastic blood film, teardrop poikilocytes and ovalocytes.

Erythroblasts can usually be found in the peripheral blood after splenectomy, and many may be present in severe anaemia and in the presence of extramedullary erythropoiesis (Fig. 5.64). Large numbers are frequently seen in the blood films of patients with sickle cell anaemia in painful crises. Small numbers of erythroblasts are not uncommon in blood from patients suffering from cyanotic heart failure or septicaemia.

Figure 5.64 Photomicrograph of a blood film. β thalassaemia major with inadequate blood transfusion support. There are numerous erythroblasts; there are also hypochromic cells, target cells and a cell containing a Howell–Jolly body.

It should be noted that when the term normoblast is used, it implies that erythroid maturation is normoblastic. Erythroblast is a more general term that also includes megaloblasts.

Effects of splenectomy and hyposplenism

Some of the effects of splenectomy and hyposplenism have already been mentioned – namely, the occurrence of target cells, acanthocytes, Howell–Jolly bodies and Pappenheimer bodies (Fig. 5.57). In addition, there may be neutrophilia (early after splenectomy), lymphocytosis, thrombocytosis and giant platelets. In people who are haematologically normal, the blood film features of hyposplenism are variable – sometimes striking and sometimes very minor.

Scanning electron microscopy

The morphology of red cells, as illustrated in this chapter, may be distorted by spreading and drying films in the traditional way. A more authentic portrayal of red cell shape in vivo can be seen by scanning electron microscopy. This provided the means for a critical re-examination of red cell morphology. Bessis and his co-workers published excellent photographs of pathological red cells and, from their appearances, proposed a terminology ^7,^21,²² that has generally been adopted in this chapter. They also discussed the difficult question of the in vivo significance of crenation (echinocytic change) observed in vitro. It seems that neither echinocytosis nor acanthocytosis is necessarily associated with increased haemolysis. It cannot be concluded, either, that crenation is occurring in vivo, when the phenomenon is markedly evident in films made on glass slides. To ensure that cells are crenated in any blood sample as it is withdrawn, Brecher and Bessis recommended that the blood be examined immediately between plastic, instead of glass coverslips or slides, to avoid the known ‘echinocytogenic’ effect of glass surfaces, probably caused by alkalinity.²² Nevertheless, for practical purposes, if a blood film of a freshly drawn blood specimen shows echinocytosis and films from other patients prepared using the same glass slides do not, the abnormality can be accepted as genuine.

The specialized procedure of scanning electron microscopy is not practical as a routine but helps in understanding the nature of cells observed in stained blood films. Morphological changes in red cells may be very complex. Echinocytic and stomatocytic change can be superimposed on other pathological forms, giving rise to ‘sickle-stomatocytes’ and ‘stomato-acanthocytes’. Acanthocytes can undergo crenation, the product being termed an ‘acantho-echinocyte’. Following splenectomy in patients with hereditary spherocytosis, sphero-acanthocytes may be observed.

The appearance of various cells by scanning electron microscopy is illustrated in Figures 5.65–5.72.

Figure 5.65 Scanning electron microscope photograph. Normal red cells.

Figure 5.66 Scanning electron microscope photograph. Hereditary spherocytosis. Note the round shape of spherocytes but also that there are cells intermediate in form between spherocytes and normal discocytes. (Compare with Fig. 5.65; also see blood film appearances as shown in Fig. 5.21.)

Figure 5.67 Scanning electron microscope photograph. Normal blood after standing overnight. Note crenation.

Figure 5.68 Scanning electron microscope photograph. Acanthocytosis. Some cells also show crenation and contraction. (Compare with Fig. 5.67; see also blood film appearances as shown in Fig. 5.45.)

Figure 5.69 Scanning electron microscope photograph. Oxidant drug-induced haemolysis; see blood film appearances shown in Figure 5.43.

Figure 5.70 Scanning electron microscope photograph. Iron deficiency anaemia. (Compare with Fig. 5.71, and see also Fig. 5.12.)

Figure 5.71 Scanning electron microscope photograph. β thalassaemia major, postsplenectomy. Shows cells grossly deficient in haemoglobin; there are also contracted cells and poikilocytes. In the hypochromic cells, inclusions are seen, corresponding to Pappenheimer bodies.

Figure 5.72 Scanning electron microscope photograph. Sickle cell anaemia (homozygosity for haemoglobin S). Shows sickled cells.

Morphology of leucocytes

This section will include a description of the normal leucocytes, some congenital anomalies and reactive changes that are commonly encountered. To describe adequately the various changes found in malignant conditions would require a lengthy text and many illustrations that are beyond the scope of this book. They will be referred to briefly here, but for detailed reference readers should consult a specialist test ²³ or an atlas on blood cells. For classification of the acute leukaemias, see the original description by the FAB (French-American-British) group²⁴ and the subsequent revision.²⁵ There is also a World Health Organization classification,²⁶ which supercedes the FAB classifications when facilities are available for fully characterizing leukaemia and is now in widespread use. However, the FAB classification remains useful as a widely accepted scheme for the initial morphologic description of leukaemias.

Polymorphonuclear neutrophils

In normal adults, neutrophils account for more than half the circulating leucocytes. They are the main defence of the body against pyogenic bacterial infections. Normal neutrophils are uniform in size, with an apparent diameter of about 13 μm on a film. They have a segmented nucleus and, when stained, pink/orange cytoplasm with fine granulation (Fig. 5.73). The majority of neutrophils have three nuclear segments (lobes) connected by tapering chromatin strands. The chromatin shows clumping and is usually condensed at the nuclear periphery. A small percentage have four lobes, and occasionally five lobes may be seen. Up to 8% of circulating neutrophils are unsegmented or partly segmented (‘band’ forms) (discussed later).

Figure 5.73 Photomicrograph of a blood film. Normal polymorphonuclear neutrophil and normal eosinophil.

In women, 2–3% of the neutrophils show an appendage at a terminal nuclear segment. This ‘drumstick’ is about 1.5 μm in diameter and is connected to the nucleus by a short stalk. It represents the inactive X chromosome and corresponds to the Barr body of buccal cells.

Occasionally, red cells will adhere to neutrophils, forming rosettes. The mechanism is unknown, but it is likely to be immune; usually it appears to be of no clinical significance but occasionally it is seen in an immune haemolytic anaemia. Leucoagglutination also occurs as an in vitro artefact.²⁷ Occasionally neutrophils (and/or monocytes) have phagocytosed erythrocytes (Fig. 5.74). This is particularly common in paroxysmal cold haemoglobinuria.

Figure 5.74 Photomicrograph of a blood film. Erythrophagocytosis in a patient with a positive direct antiglobulin test.

It is extremely important to ensure the consistency of staining of the blood films using a standardized Romanowsky method (see Chapter 4) because changes in the staining density, colour and appearance of cytoplasmic granulation, if not artefact, may have diagnostic significance. Common neutrophil abnormalities are described later in this chapter.

Granules

Toxic granulation is the term used to describe an increase in staining density and possibly number of granules that occurs regularly with bacterial infection and often with other causes of inflammation (Fig. 5.75). It can also be a feature of administration of granulocyte colony-stimulating factor (G-CSF) and of aplastic anaemia. Poorly staining (hypogranular) and agranular neutrophils occur in the myelodysplastic syndromes (Fig. 5.76) and in some forms of myeloid leukaemia.

Figure 5.75 Photomicrograph of a blood film. Severe infection. Neutrophils show toxic granulation.

Figure 5.76 Photomicrograph of a blood film. Myelodysplastic syndrome. Shows a hypogranular neutrophil and a normally granulated neutrophil.

There are rare inherited disorders that are manifest by abnormal neutrophils. In the Alder–Reilly anomaly, the granules are very large, are discrete, stain deep red and may obscure the nucleus (Fig. 5.77). Other leucocytes, including some lymphocytes, also show the abnormal granules. In the Chédiak–Higashi syndrome there are giant but scanty azurophilic granules (Fig. 5.78), and the other leucocyte types may also be affected. Alder–Reilly neutrophils function normally, but in Chédiak–Higashi syndrome there is a functional defect that is manifested by susceptibility to severe infection.

Figure 5.77 Photomicrograph of a blood film. Alder–Reilly anomaly. The nucleus is obscured by the cytoplasmic granules.

Figure 5.78 Photomicrograph of a blood film. Chédiak–Higashi syndrome. Neutrophils show abnormal granules.

Vacuoles

In blood films spread without delay, the presence of vacuoles in the neutrophils is usually indicative of severe sepsis, when toxic granulation is usually also present. Vacuoles will develop as an artefact with prolonged standing of the blood before films are made (see Chapter 1, Fig. 1.3, see p. 8).

Bacteria

Very rarely, in the presence of overwhelming septicaemia (e.g. meningococcal or pneumococcal), bacteria may be seen within vacuoles or apparently lying free in the cytoplasm of neutrophils. When blood is taken from an infected central line, clumps of bacteria or fungi may be seen scattered in the film as well as in neutrophils in phagocytic vacuoles (Fig. 5.79). In premature infants with staphylococcal septicaemia, the detection of bacteria in neutrophils helps in early diagnosis.²⁸ In endemic areas, detection of bacteria within neutrophils is important in the diagnosis of ehrlichiosis and anaplasmosis.

Figure 5.79 Photomicrograph of a blood film. Blood collected from infected site, showing bacteria (A) in scattered clumps and in a neutrophil (B).

Döhle Bodies

Döhle bodies are small, round or oval, pale blue-grey structures usually found at the periphery of the neutrophil. They consist of ribosomes and endoplasmic reticulum. They are seen in bacterial infections but also following tissue damage, in inflammation, following administration of G-CSF and during pregnancy. There is also a benign inherited condition known as May–Hegglin anomaly with a similar but not identical morphologic structure; in this condition, the inclusions occur in all types of leucocytes except lymphocytes.

Nuclei

Segmentation of the nucleus of the neutrophil is a normal event as the cell matures from the myelocyte. With the three-lobed neutrophil as a marker, a shift to the left (less mature) or to the right (hypermature) can be recognized (Table 5.2). A left shift with band forms, metamyelocytes and perhaps occasional myelocytes is common in sepsis (Fig. 5.80), when it is usually accompanied by toxic granulation. If promyelocytes and myeloblasts are also present, it is likely to be a feature of a leucoerythroblastic anaemia or leukaemia (Fig. 5.81); occasionally this extreme picture may be seen in very severe infections, when it is called ‘leukaemoid reaction’. A left shift, with a significant number of band forms, occurs normally in pregnancy.

Table 5.2 Stages of granulocyte maturation

Figure 5.80 Photomicrograph of a blood film. Infection. Shows left shift of the neutrophils with toxic granulation.

Figure 5.81 Photomicrograph of a blood film. Chronic myelogenous leukaemia. There is a left shift with band forms, metamyelocytes, myelocytes and one myeloblast.

Hypersegmentation

The presence of hypersegmented neutrophils is an important diagnostic feature of megaloblastic anaemias. Neutrophil hypersegmentation can be defined as the presence of neutrophils with six or more lobes or the presence of more than 3% of neutrophils with at least five lobes. In florid megaloblastic states, neutrophils are often enlarged and their nuclei may have six or more segments connected by particularly fine chromatin bridges (Fig. 5.10). A right shift with moderately hypersegmented neutrophils may also be seen in uraemia and not infrequently in iron deficiency.²⁹ Hypersegmentation can be seen after cytotoxic treatment, especially with methotrexate. Patients undergoing treatment with hydroxycarbamide or other drugs that induce megaloblastosis sometimes develop hypersegmented neutrophils (Table 5.2).³⁰

Pelger–Huët Cells

The Pelger–Huët anomaly is a benign inherited condition in which neutrophil nuclei fail to segment properly. The majority of circulating neutrophils have only two discrete equal-sized lobes connected by a thin chromatin bridge (Fig. 5.82). The chromatin is coarsely clumped, and granule content is normal.

Figure 5.82 Photomicrograph of a blood film. Pelger–Huët anomaly. Shows hypolobated neutrophils.

Figure 5.83 Photomicrograph of a blood film. Chronic myelogenous leukaemia. There are five ‘pseudo-Pelger’ cells; this abnormality is not seen in the chronic phase of this disease.

Pyknotic Neutrophils (Apoptosis)

Small numbers of dead or dying cells may normally be found in the blood, especially when there is an infection. They may also develop in normal blood in vitro after standing for 12–18 h, even if kept at 4°C. These cells have round, dense, featureless nuclei and their cytoplasm tends to be dark pink (see p. 8 and Fig. 1.4). It is important not to confuse these cells with erythroblasts.

Eosinophils

Eosinophils are a little larger than neutrophils, 12–17 μm in diameter. They usually have two nuclear lobes or segments, and the cytoplasm is packed with distinctive spherical gold/orange (eosinophilic) granules (Figs 5.73, 5.84). The underlying cytoplasm, which is usually obscured by the granules, is pale blue. Prolonged steroid administration causes eosinopenia. Moderate eosinophilia occurs in allergic conditions; more severe eosinophilia (20–50 × 10⁹/l) may be seen in parasitic infections and even greater numbers may be seen in other reactive eosinophilias, eosinophilic leukaemia and the idiopathic hypereosinophilic syndrome. Reactive eosinophilia with very high counts may be seen in T-cell lymphoma, B-cell lymphoma and acute lymphoblastic leukaemia. Eosinophils are part of the leukaemic population in chronic myelogenous leukaemia, and this is occasionally so in acute myeloid leukaemia. Cytological abnormalities in eosinophils are not very useful in distinguishing between leukaemic and reactive eosinophils.

Figure 5.84 Photomicrograph of a blood film. Normal adult. Shows a basophil, an eosinophil and a neutrophil.

Basophils

Basophils are the rarest (<1%) of the circulating leucocytes. Their nuclear segments tend to fold up on each other, resulting in a compact irregular dense nucleus resembling a closed lotus flower. The distinctive, large, variably sized, dark blue or purple granules of the cytoplasm (Fig. 5.84) often obscure the nucleus; they are rich in histamine, serotonin and heparin. Basophils tend to degranulate, leaving cytoplasmic vacuoles.

Basophils are present in increased numbers in myeloproliferative neoplasms and are especially prominent in chronic myelogenous leukaemia; in the latter condition, when basophils are more than 10% of the differential leucocyte count, this is a sign of impending accelerated phase or blast crisis.

Monocytes

Monocytes are the largest of the circulating leucocytes, 15–18 μm in diameter. They have bluish-grey cytoplasm that contains variable numbers of fine reddish granules. The nucleus is large and curved, often in the shape of a horseshoe, but it may be folded or curled (Figs 5.85, 5.86). It does not undergo segmentation. The chromatin is finer and more evenly distributed than in neutrophil nuclei. An increased number of monocytes occur in some chronic infections and inflammatory conditions such as tuberculosis and Crohn’s disease, in chronic myeloid leukaemias (particularly atypical chronic myeloid leukaemia and chronic myelomonocytic leukaemia) and in acute leukaemias with a monocytic component. In chronic myelomonocytic leukaemia, the mature monocyte count may reach as high as 100 × 10⁹/l. It is occasionally difficult to distinguish abnormal monocytes from the large activated T lymphocytes produced in infectious mononucleosis or from circulating high-grade lymphoma cells.

Figure 5.85 Photomicrograph of a blood film. Healthy adult. Monocyte.

Figure 5.86 Photomicrograph of a blood film. Healthy adult. Shows a monocyte and a lymphocyte.

Lymphocytes

The majority of circulating lymphocytes are small cells with a thin rim of cytoplasm, occasionally containing scanty azurophilic granules (Figs 5.1, 5.87). Nuclei are remarkably uniform in size (about 9 μm in diameter). This provides a useful guide for estimating red cell size (normally about 7–8 μm) on the blood film. Some 10% of circulating lymphocytes are larger, with more abundant pale blue cytoplasm containing azurophilic granules (Fig. 5.87). The nuclei of lymphocytes have homogeneous chromatin with some clumping at the nuclear periphery. About 85% of the circulating lymphocytes are T cells or natural killer (NK) cells.

Figure 5.87 Photomicrograph of a blood film. Shows a small lymphocyte and two large granular lymphocytes with azurophilic granules.

In infections, both bacterial and viral, transformed lymphocytes may be present. These immunoblasts or ‘Türk’ cells are 10–15 μm in diameter, with a round nucleus, often with a large nucleolus, and abundant, deeply basophilic cytoplasm (Fig. 5.88). They may develop into plasmacytoid lymphocytes and plasma cells, and these are occasionally seen in the blood in severe infections. In the absence of infection, multiple myeloma must be excluded. In viral infection, ‘reactive lymphocytes’ appear in the blood. These have slightly larger nuclei with more open chromatin and abundant cytoplasm that may be irregular. The most extreme examples of these cells are usually found in infectious mononucleosis (Fig. 5.89). These ‘glandular fever’ cells have irregular nuclei and abundant cytoplasm that is basophilic at the periphery; they have a tendency to appear, on a blood film, to have flowed around adjacent erythrocytes.

Figure 5.88 Photomicrograph of a blood film. Viral infection. Shows a Türk cell, another reactive lymphocyte and a small lymphocyte.

Figure 5.89 Photomicrograph of a blood film. Infectious mononucleosis. There are two activated lymphocytes (‘atypical mononuclear cells’).

Malignant lymphoid cells vary enormously in their morphology. The commonest malignancy is chronic lymphocytic leukaemia, the leukaemic population being composed almost exclusively of small lymphocytes (Fig. 5.90), sometimes with a few larger nucleolated cells. In prolymphocytic leukaemia, the majority of cells are a little larger than small lymphocytes with more cytoplasm and usually one distinct nucleolus (Fig. 5.91). Lymphoblasts of acute lymphoblastic leukaemia (Figs 5.92, 5.93) vary in size from only slightly larger than lymphocytes to cells of 15–17 μm diameter. The nuclei generally have diffuse chromatin, but there may be some chromatin condensation in the smaller blasts. The cytoplasm varies from weakly to strongly basophilic.

Figure 5.90 Photomicrograph of a blood film. Chronic lymphocytic leukaemia. The cells are small lymphocytes; note that rouleaux formation is increased.

Figure 5.91 Photomicrograph of a blood film. Prolymphocytic leukaemia. There is a uniform population of prolymphocytes.

Figure 5.92 Photomicrograph of a blood film. Philadelphia-positive acute lymphoblastic leukaemia (FAB L1 category). Shows three lymphoblasts.

Figure 5.93 Photomicrograph of a blood film. Acute lymphoblastic leukaemia (FAB L1 type). Shows lymphoblasts.

Circulating lymphoma cells vary markedly in size, depending on the type of lymphoma. When there is a lymphocytosis, the lymphocytes are usually far less uniform than in chronic lymphocytic leukaemia, and the lymphoma cells frequently have irregular lobed, indented or cleaved nuclei and relatively scanty agranular cytoplasm that varies in its degree of basophilia. Lobulated lymphocytes are a feature of HTLV-I (human T-lymphotropic virus type I) infection and of adult T-cell leukaemia/lymphoma. However, lymphocytes with definite lobulation are also a common storage artefact in blood kept for 18–24 h at room temperature (see p. 7).

Lymphocytes predominate in the blood films of infants and young children. In this age range, large lymphocytes and reactive lymphocytes tend to be conspicuous, and a small number of lymphoblasts may also be present.

There is no uniform approach to the terminology used to refer to cytologically abnormal lymphocytes, a problem compounded by the fact that sometimes it is difficult to distinguish reactive lymphocytes from neoplastic. The term ‘variant lymphocyte’ is sometimes used but it is not a term that is known to many clinicians. It is not prudent to describe lymphocytes as ‘reactive’; it is better to refer to ‘atypical lymphocytes, appear reactive’ or, in other circumstances, ‘abnormal lymphocytes, suspect lymphoma’. In this way an opinion, which may be wrong, is differentiated from a factual morphologic description.

Platelet morphology

Normal platelets are 1–3 μm in diameter. They are irregular in outline with fine red granules that may be scattered or centralized. A small number of larger platelets, up to 5 μm in diameter, may be seen in normal films. Larger platelets are seen in the blood when platelet production is increased (Fig. 5.94) and in hyposplenism (Fig. 5.95). Thus, for example, in severe immune thrombocytopenia, some large platelets will be seen on the film. Very high platelet counts as a feature of a myeloproliferative neoplasm may be associated with extreme platelet anisocytosis, with some platelets being as large as red cells and often with some agranular or hypogranular platelets (Fig. 5.96). The platelet count frequently increases with acute inflammatory stress or bleeding but seldom to more than 1000 × 10⁹/l. More than this, unless the patient is critically ill or hyposplenic, makes a myeloproliferative neoplasm a definite possibility.

Figure 5.94 Photomicrograph of a blood film. Essential thrombocythaemia. Shows platelet anisocytosis and increased numbers of platelets.

Figure 5.95 Photomicrograph of a blood film. Hyposplenism in coeliac disease. Shows giant platelet.

Figure 5.96 Photomicrograph of a blood film. Myeloproliferative neoplasm. Shows platelet anisocytosis with some giant platelets.

Characteristic morphologic features are seen in various uncommon inherited platelet disorders associated with bleeding. These include the Bernard–Soulier syndrome, in which there are giant platelets with a defective ristocetin response, and the grey platelet syndrome, in which the platelets have decreased granules and have a ghost-like appearance on the stained blood film (Fig. 5.97). Thrombocytopenia with large platelets is also a feature of the May–Hegglin anomaly (see p. 92).

Figure 5.97 Photomicrograph of a blood film. Grey platelet syndrome. Shows severely hypogranular platelets.

In about 1% of individuals, ethylenediaminetetra-acetic acid (EDTA) anticoagulant causes platelet clumping, resulting in pseudothrombocytopenia.³¹ It is much less likely to occur when the blood is collected into any other anticoagulant.³² This phenomenon may be detected when it gives rise to a ‘flag’ on an automated blood cell counter; it is identifiable on the blood film. It is not associated with any coagulation disturbance and platelet function is normal. When this abnormality is detected it is important that the erroneous platelet count is deleted from the report. If the blood film shows that the platelet count is clearly normal, the statement ‘Platelet count normal’ is sufficient, rather than a repeat sample in an alternative anticoagulant being requested. Occasionally, EDTA inhibits the staining of platelets.³³

Occasionally, platelets may be seen adhering to neutrophils (Fig. 5.98).³⁴^–³⁷ This has been reported in patients who have demonstrable antiplatelet autoantibodies,³⁸ but it is more commonly seen in apparently healthy individuals. It is not seen in films made directly from blood that has not been anticoagulated. Sometimes the platelets are ingested by neutrophils.³⁹ If a blood film in a patient with platelet satellitism or phagocytosis shows that the automated count is erroneous and the platelet count is in fact normal, the erroneous count should be deleted from the report and an explanation given.