4. The leukocytes Leukocytes, commonly called ‘white blood cells’ (WBC), possess a nucleus, which allows them to be easily distinguished from anucleated mature erythrocytes. Typically, five types of leukocytes circulate in the peripheral blood of mammals: neutrophils, eosinophils, basophils, lymphocytes and monocytes. Broadly, leukocytes are responsible for controlling and effecting inflammation to provide tissue ‘defence’. Each type of leukocyte has specialised functions; some have phagocytic and microbicidal activity, while others produce the cytokines that mediate the inflammatory process. The main function of neutrophils is phagocytosis; that is, to ingest and wherever possible ‘kill’ and breakdown the ingested particle, using a combination of stored proteins and cytotoxic substances generated in an oxygen-dependent process. Eosinophils also have phagocytic activity and may be involved in any inflammatory process, but are usually most abundant at sites of inflammation with an allergic or parasitic aetiology, where they have roles in the regulation of hypersensitivity in the former and as cytotoxic effector cells in the latter. Basophils are usually present in the least concentration of any leukocyte in the peripheral blood and have the least understood function, with postulated roles in allergic conditions, haemostasis, lipid metabolism and cytotoxicity. Basophils have a number of functions in common with tissue mast cells, but develop from different precursor cells. Lymphocytes have several complex roles in the regulating and effecting of immune and inflammatory processes. Some subsets of lymphocytes (CD4+ T cells) produce cytokines that regulate the function of leukocytes whereas others are effectors, mediating cytotoxicity (CD8+ T cells, natural killer (NK) cells) or producing antibodies (plasma cells). The mononuclear phagocytic cells or macrophages present in tissues largely originate from monocytes in the peripheral blood and, as their name suggests, have a role in the phagocytosis and breakdown of material. These cells also have an important role in the production of the cytokines that regulate inflammatory process. The leukocytes that are found in the peripheral blood of mammals are in transit between the sites of production (predominantly the bone marrow in mature animals) and the tissues. Changes in the concentration of these transitory leukocytes may reflect tissue demand and/or bone marrow production, and can be used to identify the disease processes. This chapter describes the general characteristics of leukocytes encountered in the peripheral blood of Australian mammals. The specific characteristics of leukocytes for individual species, where known, are reported in Chapter 9. Leukocytes are classified according to their nuclear and cytoplasmic characteristics. Neutrophils, eosinophils and basophils are collectively referred to as granulocytes. These all possess a nucleus that is divided into several lobes by ‘constrictions’ in the width of the nucleus. The staining characteristics of the secondary (or ‘specific’) granules in the cytoplasm are used to differentiate between the three types of granulocytes. Lymphocytes and monocytes are referred to as mononuclear cells and typically possess a nucleus without segmentation. The proportion and absolute concentration of each type of leukocyte may vary between species and between individuals of the same species. Neutrophils In many species, neutrophils are the most commonly observed granulocyte in the peripheral blood. They typically have a nucleus with 3–6 lobes, composed of coarsely clumped chromatin contained by a nuclear membrane that usually has a ‘rough’ appearance in mature cells and ‘smooth’ in immature cells. Neutrophils have a moderate amount of colourless cytoplasm that contains granules, both primary (azurophilic) and secondary (specific), which are usually not apparent (Figure 4.1). Under some circumstances these granules may stain positively, in which case they appear as many small, fine granules that are either mildly eosinophilic or basophilic and are evenly distributed throughout the cytoplasm. If the secondary granules are consistently prominent after staining, the term ‘heterophil’ is used instead of neutrophil. This is the case with some Australian mammals, such as the dugong (see p. 146). The size of neutrophils has been reported as 6–9 µm for several species of macropodids, but larger (10–13 µm) in the yellow-footed rock-wallaby, 15 µm in the common wombat (Ponder et al., 1928), 9.2–16.8 µm in the common brushtail possum (Barbour, 1972), 9.6–14.4 µm in the platypus (Canfield and Whittington, 1983) and 12.6–15 µm in the koala (Canfield and Dickens, 1982).
INTRODUCTION
MORPHOLOGICAL APPEARANCE OF LEUKOCYTES
No. of lobes | ||
1 (band) | 0 | 0 |
2 | 0–1 | 0–1 |
3 | 1–8 | 2–8 |
4 | 21–48 | 20–38 |
5 | 38–63 | 28–50 |
6 | 3–21 | 10–28 |
7 | 0–7 | 4–12 |
1 Clark unpublished data
2 Clark et al., 2002
The nucleus of cells that represent the penultimate stage of neutrophil maturation, known as a ‘band’ neutrophils, have a less segmented nucleus than mature neutrophils, defined as a nucleus that does not have any constrictions greater than 50% of its width. The nucleus of these cells usually also has a smooth membrane and more homogeneous chromatin than mature neutrophils. Band neutrophils are generally infrequently observed in the peripheral blood of healthy animals. Less mature stages of neutrophil development may be observed in the bone marrow (and other haematopoietic tissues) and very infrequently in the blood of most healthy animals. An exception is the platypus, in which there may be band neutrophils and less mature stages of neutrophil development in the peripheral blood of clinically healthy animals (Whittington and Grant, 1983, 1984).
As neutrophils age, the number of nuclear lobes typically increases and aged (senescent) neutrophils become ‘hyper-segmented’. In most domestic animal species, more than five nuclear lobes indicate that a cell is senescent (Jain, 1986). However, in many species of marsupials and rodents, neutrophils commonly contain five or more nuclear lobes, making the determination of senescence difficult in most circumstances (Hawkey, 1977). The number of nuclear lobes and the distribution of the respective neutrophils for clinically healthy Tammar wallabies (Clark et al., 2002b) and eastern grey kangaroos (Clark, unpublished data) is given in Table 4.1. In both species, few band neutrophils are evident, the majority of cells have 4–6 nuclear lobes and neutrophils with seven nuclear lobes are not uncommon.
Eosinophils
Eosinophils typically have a nucleus with 1–3 lobes that are composed of coarsely clumped chromatin (but usually less dense than neutrophils) (Figure 4.2). The cytoplasm of eosinophils, when apparent, is usually more basophilic than that of neutrophils. However, the characteristics of the cytoplasm are usually obscured by the presence of secondary granules, which are eosinophilic and vary in size, shape, density and hue between species. For example, the eosinophils of the common brushtail possum contain regular, rod-shaped, brightly eosinophilic, densely packed granules whereas those from swamp wallabies have large, brightly eosinophilic ovoid granules throughout the cytoplasm, and the eosinophils from New Zealand sea-lions have small, round, eosinophilic to brown granules that give a dusty appearance to the cytoplasm. Within a species the granules are usually uniform. An exception is the dingo, for which the size, number and density of granules may vary between eosinophils within the same individual and between different individuals.
The size of eosinophils, measured by light microscopy, has been reported as 7–8 µm for several species of macropodids, 11 µm in the yellow-footed rock-wallaby, 13–15 µm in the common wombat (Ponder et al., 1928), 10.2–16.8 µm in the common brushtail possum (Barbour, 1972), 13.2–16.8 µm in the platypus (Canfield and Whittington, 1983) and 12.6–17.4 µm in the koala (Canfield and Dickens, 1982).
The number of eosinophils observed in health varies between species. Eosinophils are regularly observed in most species of macropodids, but are very rarely observed in some species of dunnart (Haynes and Skid-more, 1991).
Basophils
The striking characteristic of the basophil is the basophilic secondary granules in the cytoplasm (Figure 4.3). The number and size of these granules varies between species, but typically they are round, intensely basophilic and present at high density within the cytoplasm, to the extent that the nucleus may be obscured. The eastern barred bandicoot and eastern grey kangaroo are examples of species that possess basophils that conform to this description. In contrast, the basophils of the New Zealand fur-seal contain only a few, small, sparsely distributed, basophilic granules. When the nucleus of a basophil is visible, it typically has 3–5 lobes composed of coarsely clumped chromatin. If apparent, the cytoplasm usually has a pale basophilic hue.
Plate 1 Mesothelial cells, probably originating from the pericardium, in a sample of blood collected by cardiac puncture from a common ringtail possum. These were present at the ‘leading edge’ of the blood film. (WG stain.)
Plate 2 Commercially available tubes that contain EDTA (the preferred anticoagulant for haematological assessment). The tube should be the appropriate size for the volume of blood to be collected.
Plate 3 Four blood films, each with some individual characteristics. All have intact cells and regions of cell density that allow adequate visualisation of cells (i.e. a ‘monolayer’).
Plate 4 Lysed leukocytes (‘smudge’ cells), in a blood film from a brush-tailed phascogale, should not be interpreted (MGG stain).
Plate 5 Fungal growth on a blood film. The contamination occurred prior to processing (as the organism is stained) and is most commonly encountered with delayed fixation and staining. (MGG stain.)
Plate 6 Blood from a common wombat. Many of the erythrocytes exhibit multiple, small, refractile structures (a) or a pale, ‘moth-eaten’ appearance (b). Both of these artefacts are caused by incomplete drying of the slide prior to staining. (MGG stain.)
Plate 7 Stain precipitate in a blood film from a brush-tailed phascogale, characterised by purplish to basophilic, flocculent material present both on cells and in the background. Care must be taken not to misinterpret small amounts of precipitated stain on erythrocytes as haemoparasites. (DQ stain.)
Plate 8 Poorly stained haematological cells from a mountain brushtail possum. This may occur with fixation of blood films by formalin vapours. (MGG stain.)
Plate 9 Anucleated squamous epithelial cells in a sample of blood collected from an agile antechinus. These originated from the skin and may have adherent bacteria. (MGG stain.)
Plate 10 A cluster of epithelial cells, consistent with salivary gland origin, in the blood of a yellow-footed rock-wallaby. The sample was collected by venepuncture of the jugular vein, which in macropodids is covered by a lobe of the parotid salivary gland. (MGG stain.)
Plate 11 Powder from the disposable gloves worn by the operator in a blood sample from a New Zealand fur-seal. These should not be mistaken for organisms. (MGG stain.)
Plate 12 Typical mammalian erythrocytes showing a pale central region and mild anisocytosis (antilopine wallaroo; WG stain).
Plate 13 Erythrocytes from a short-finned pilot whale. Note the absence of central pallor. (MGG stain.)
Plate 14 Transmission electron micrograph of erythrocytes from a brush-tailed rock-wallaby (LCUA stain).
Plate 15 Scanning electron micrograph of erythrocytes from a Tammar wallaby showing typical biconcave shape.
Plate 16 Scanning electron micrograph of erythrocytes from a pygmy sperm whale that are ‘cup-shaped’.
Plate 17 Blood from a Gould’s wattled bat showing four polychromatophilic erythrocytes (arrow) amongst mature erythrocytes (WG stain).
Plate 18 Reticulocyte (arrow) in the blood of a Parma wallaby (NMB stain).
Plate 19 Two nucleated erythrocytes (metarubricytes) in a blood sample from a clinically healthy swamp wallaby. Note also the single polychromatophilic erythrocyte (arrow) and rouleaux of erythrocytes. (MGG stain.)
Plate 20 Blood film from a stripe-faced dunnart in which most of the erythrocytes are echinocytes, most likely caused by the osmotic effect of the anticoagulant (EDTA) (MGG stain).
Plate 21 Scanning electron micrograph of erythrocytes from a New Zealand fur-seal in which most of the cells are echinocytes.
Plate 22 Blood film from a koala in which several of the erythrocytes are stomatocytes, one of which has basophilic stippling (arrow) (MGG stain).
Plate 23 Blood film from a clinically healthy Parma wallaby in which most of the erythrocytes are codocytes (‘target cells’) (MGG stain).
Plate 24 Keratocyte (a) and Heinz body (b) in a blood film from a common brushtail possum infected with ‘wobbly possum disease’ virus (DQ stain).
Plate 25 A ‘blister cell’ (arrow) in a blood sample from a common brushtail possum infected with ‘wobbly possum disease’ virus (DQ stain).
Plate 26 Blood sample from a dingo in which most of the erythrocytes are torocytes, which represent an artefactual redistribution of haemoglobin and should not be confused with hypochromatic erythrocytes (MGG stain).
Plate 27 Spherocyte (arrow) in the blood film of a spotted-tailed quoll (W stain).
Plate 28 Scanning electron micrograph of erythrocytes from a brush-tailed rock-wallaby showing several discocytes, a knizocyte (arrow) and a cell lacking the central depression.
Plate 29 Howell-Jolly body (arrow) in an erythrocyte from a Parma wallaby (MGG stain).
Plate 30 Capillary tubes containing blood after centrifugation that illustrate the packed cell volume of three bilbies: two animals had a value of 0.38 L/L and the third had a lesser value of 0.30 L/L.
Plate 31 A blood film from an anaemic dusky antechinus, in which most of the erythrocytes are polychromatophilic, which indicates a ‘regenerative’ response (WG stain).
Plate 32 Blood film from an anaemic common brushtail possum illustrating an attempted regenerative response. Polychromatophilic erythrocytes and stages of nucleated erythrocyte development (metarubricytes and rubricytes) and a lymphocyte (arrow) can be seen. (MGG stain.)
Plate 33 A Lymphocyte with an indented nucleus and one with a cleaved nucleus (arrow) in the blood from an eastern barred bandicoot (WG stain).
Plate 34 Lymphocyte from a Parma wallaby with an indented (‘reniform’) nucleus and several azurophilic granules (MGG stain).
Plate 35 Annular leukocyte from an eastern barred bandicoot, showing a thick nuclear ‘ring’ and one slight constriction. The cytoplasm is basophilic and lacks apparent secondary granules, suggesting a monocytic or neutrophilic lineage. (MGG stain.)
Plate 36 Annular leukocyte from a northern brown bandicoot with a nucleus composed of several lobes, one separated by fine strands of chromatin. The many eosinophilic granules present in the cytoplasm identify the cell’s lineage. (DQ stain.)
Plate 37 Transmission electron micrograph of a neutrophil from a New Zealand sea-lion (LCUA stain, original magnification ×4,380).
Plate 38 Transmission electron micrograph of an eosinophil from a common brushtail possum (LCUA stain, original magnification ×4,380). (Courtesy of M. Cooke, Massey University.)
Plate 39 Transmission electron micrograph of a lymphocyte from a Tammar wallaby (LCUA stain, original magnification ×4,380).
Plate 40 Transmission electron micrograph of a lymphocyte from a brush-tailed rock-wallaby. A prominent nucleolus is evident in the nucleus. (LCUA stain, original magnificatio 4,380.)
Plate 41 Transmission electron micrograph of a monocyte from a brush-tailed rock wallaby (LCUA stain, original magnification ×4,380).
Plate 42 Neutrophil from a common brushtail possum stained with chloroacetate esterase, a cytochemical stain (reprinted with permission from the Australian Veterinary Journal 77, 605).
Plate 43 Lymphocytes in the peripheral blood of a short-beaked echidna. Two nuclei without cytoplasm are present (arrows). (WG stain.)
Plate 44 Leukocyte from a bilby with a fragmented nucleus (karyorrhexis) (WG stain).
Plate 45 Prominent staining of the secondary granules of a neutrophil from an eastern grey kangaroo. These should not be mistaken for toxic granulation. (WG stain.)
Plate 46 Neutrophil from a sub-Antarctic fur-seal containing two Döhle bodies (arrows) (MGG stain).
Plate 47 Immature (‘band’) neutrophils from a short-beaked echidna with a fractured beak. The cytoplasm of these cells contains basophilic aggregations that represent RNA. (DQ stain.)
Plate 48 Neutrophils from the blood of a Matschie’s tree kangaroo with a neutrophilia. The cytoplasm of these cells has a pale basophilic appearance. (WG stain.)
Plate 49 Band neutrophil from a spectacled hare-wallaby showing increased cytoplasmic basophilia and vacuoles (WG stain.)
Plate 50 Band neutrophil from a common ringtail possum that had numerous bite injuries. The cell’s cytoplasm has an increased basophilic colour and a foamy appearance. (WG stain.)
Plate 51 Band neutrophil from the blood of a numbat showing a hypo-segmented nucleus and markedly basophilic cytoplasm (WG stain.)
Plate 52 Band neutrophil from the blood of a numbat showing a hypo-segmented nucleus, markedly basophilic cytoplasm and toxic granulation (WG stain.)
Plate 53 A myelocyte, metamyelocyte and neutrophil in the blood of a platypus. All these stages of myeloid development may be encountered in clinically healthy animals and do not necessarily represent a response to marked inflammation as they do in other species (MGG stain.)
Plate 54 Blood from a common ringtail possum contains many bacteria: some have been phagocytosed by neutrophils, others are ‘free’ in the blood (MGG stain.)
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