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.

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.

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

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