6. Haematopoiesis

INTRODUCTION

CHARACTERISTICS OF HAEMATOPOIESIS

The predominant site of haematopoiesis varies with the age of the animal. In foetal and neonatal animals the yolk sac, liver, spleen and bone marrow may all be sites of haematopoiesis. In adult mammals, the primary site of haematopoiesis is usually the bone marrow (Jain, 1986). Active haematopoietic tissue (‘red marrow’) is located in the trabecular bone at the ends of ‘long’ bones, such as the femur (Plate 61) and humerus, and ‘flat’ bones, such as the ribs and sternum. Inactive marrow, which contains a large proportion of adipocytes, and may appear creamy-yellow in colour (‘yellow marrow’), is usually observed in the shafts of long bones of mature animals.

Several studies have assessed the development of haematopoiesis of Australian mammals. In the Tammar wallaby the predominant site of haematopoiesis prior to birth is the yolk sac (Basden et al., 1996). Following birth, the liver (day 2–100), spleen (day 8–200) and bone marrow (day 10 onwards) are all sites of haematopoiesis in the quokka (Yadav, 1972). Similarly, the liver is the predominant site of haematopoiesis from birth to 14 days of age in the neonatal Tammar wallaby (Basden et al., 1996). Two weeks after birth, foci of haematopoiesis appear in the bone marrow and by day 30 the bone marrow is the major site of haematopoiesis. The liver also displays haematopoietic activity for the first 4 weeks of pouch life of the northern brown bandicoot (Cisternas and Armati, 1999).

Haematopoiesis may also occur in sites outside the bone marrow in adult animals (Plate 62). This ‘extra-medullary’ haematopoiesis has been reported in the spleen of the sugar glider (erythroid), eastern pygmy-possum, kowari, common planigale and little red antechinus (myeloid) (Canfield et al., 1990a, 1990b). Extra-medullary haematopoiesis is a common finding in laboratory rodents. The splenic red pulp is an active haematopoietic site throughout life in the mouse and splenic haematopoiesis may be observed in the adult rat; however, prominent haematopoiesis usually represents an underlying disease state (Percy and Barthold, 1993).

Haematopoiesis has been observed in the spleen and in the bone marrow of the platypus (Tanaka et al., 1988). Those authors suggested that the spleen is the primary haematopoietic organ in the platypus, as the number of proliferating haematopoietic elements within a unit area of tissue was greater in the spleen than in the bone marrow and some haematopoietic elements, such as megakaryocytes, were not present in the bone marrow. However, only one animal was assessed in their study.

Lymphocytes deserve special consideration because lymphopoiesis occurs in a number of tissues, including the thymus, lymph nodes, spleen and gut-associated lymphoid tissue (GALT), as well as in the bone marrow. The predominant site of lymphopoiesis may change during development and some tissues are required for the developmental stages of specific subsets of lymphocytes, for example, the thymus is the organ where T lymphocytes are selected for self-tolerance.

The development of lymphoid tissue has been studied in several species of Australian mammals. At birth, quokkas possess functional lymphopoietic tissue in the liver only (Ashman and Papadimitriou, 1975). In studies of this species, large lymphocytes first appear in the cervical thymus at 2 days after birth and in the thoracic thymus at 4 days after birth. Small lymphocytes appear in these sites 1–2 days later. The histological development of the two glands is similar, but the thoracic thymus develops much more slowly. The appearance of Hassall’s corpuscles in both thymus glands correlates with the onset of humoral immune responses. Lymph nodes first appear as aggregates of lymphocytes around the lymphatic vessels at 5 days of age, and then differentiate into cortex and medulla at approximately 14 days, but do not develop germinal centres until almost 90 days. Small lymphocytes are not observed in the spleen until the second week after birth, and reactive centres do not appear until after 90 days of pouch life. Peyer’s patches, lymphoid tissue present in the mucosa and submucosa of the small intestine, are not found until 60 days of age. Large lymphocytes are seen in the bone marrow at 14 days, but small lymphocytes are not found until after the first month.

A study of lymphoid tissues (i.e. cervical and thoracic thymus, lymph nodes and GALT) from birth to maturity in the Tammar wallaby showed similar development to the quokka (Basden et al., 1997). Lymphocytes were first detected in the cervical thymus at 2 days after birth and in the thoracic thymus at 6 days. Hassall’s corpuscles were apparent in the cervical thymus by 21 days and in the thoracic thymus by 30 days post partum. Lymphocytes were first detected in the lymph nodes and spleen at 4 and 12 days, respectively. However, germinal centres in the lymph nodes were not recognised until day 90, which coincided with increased immunoglobulin G concentration.

The development and morphological characteristics of the spleen, thymus, lymph nodes and liver of the northern brown bandicoot have been studied (Cisternas and Armati, 1999). Lymphopoiesis was evident in the thymus in the first week of pouch life. The spleen matured more slowly, but differentiated and showed signs of immunocompetency by the time the young left the pouch.

Cells of the bone marrow

The bone marrow contains many morphologically recognisable types and developmental stages of cells, which may be broadly classified into erythrocytic, granulocytic, monocytic, lymphocytic, megakaryocytic, and ‘additional’ cells. These are listed in Table 6.1 and selected cell types are illustrated in Plates 63–71.

Table 6.1 Morphologically recognisable developmental stages of the cells of the bone marrow

The morphological characteristics of the cells in the bone marrow, when stained with Romanowsky stains and viewed by light microscopy, have been described for a number of species of mammals and these general characteristics are presented in the following text. In addition to the morphologically recognisable cells, also present in haematopoietic tissue are a range of stem cells and progenitor cells (e.g. blast forming units and colony forming units) that cannot be morphologically distinguished (Gasper, 2000), but it is beyond the scope of this book to discuss them.

Erythrocytic cells

The morphologically recognisable stages of erythroid cell development include:

•  rubriblast

•  prorubricyte

•  rubricyte

•  metarubricyte

•  polychromatophilic erythrocyte

•  mature erythrocyte

(see Plates 65–67 for examples of selected cells types).

The rubriblast is the least mature erythroid cell that may be morphologically identified using Romanowsky stains. It has a round nucleus with a smooth nuclear membrane and dense, coarse chromatin with 1–2 pale, round, prominent nucleoli. The cytoplasm is intensely basophilic and forms a small rim around the nucleus, giving a high nuclear to cytoplasmic ratio. The prorubricyte is the next stage of erythroid maturation. Prorubricytes have a round nucleus with a smooth nuclear border and chromatin that is slightly coarser than that of the rubriblast and they lack nucleoli (as do all the following, more mature stages of erythroid development). There is an increased amount of cytoplasm (resulting in a decreased nuclear to cytoplasmic ratio), which is slightly less basophilic and may exhibit a perinuclear clear zone. These cells mature to rubricytes. Rubricytes have a smaller nucleus than the prorubricyte, with very coarse chromatin. The cytoplasm is basophilic, but may develop an eosinophilic undertone, giving an amphophilic appearance. The nuclear to cytoplasmic ratio is decreased compared with previous stages. Metarubricytes are the last nucleated stage of erythroid development. The nucleus in these cells is very dense and pyknotic and the cytoplasm is basophilic, amphophilic or eosinophilic (depending on the amount of RNA and haemoglobin present).

The penultimate stage of erythroid development is the polychromatophilic erythrocyte. These anucleated erythroid cells have a mild basophilic colour when stained with Romanowsky stains because of the presence of ribosomal RNA. Mature erythrocytes lack this RNA and have a classically eosinophilic colour because of the large amounts of haemoglobin (as previously described in Chapter 2).

Granulocytic cells

The developmental stages for each of the three types of granulocytes (i.e. neutrophils, eosinophils and basophils) include:

•  myeloblast

•  promyelocyte

•  myelocyte

•  metamyelocyte

•  band granulocyte

•  mature granulocyte

(see Plates 66–70 for examples of selected cells types).

Myeloblasts are the least mature stage of myeloid cell development that may be recognised in Romanowsky-stained samples. They are characterised by a large round to irregular nucleus with fine chromatin and one or more prominent, round nucleoli. The cytoplasm is a pale basophilic colour and generally does not contain visible granules. The promyelocyte is the next stage of myeloid development. These cells are a similar size to myeloblasts, but have an increased amount of cytoplasm, which contains small azurophilic granules. The nucleus is similar in appearance to myeloblasts, being round to irregular in shape with fine chromatin, but generally lacks visible nucleoli. Promyelocytes mature to the next stage of development, the myelocyte. Myelocytes have a round to ovoid nucleus with fine to reticular chromatin and lack visible nucleoli. The cytoplasm contains visible secondary granules and cells can be identified as belonging to the neutrophil, eosinophil or basophil series. As the cells develop, the nucleus becomes indented or ‘kidney-shaped’, and the cell is referred to as a metamyelocyte. The nucleus is composed of reticular to coarse chromatin and a moderate amount of cytoplasm that contains the appropriate secondary granules for the granulocyte line. In the bone marrow of marsupials, there may be leukocytes with an annular nucleus, which correspond to the metamyelocyte stage of development (Plates 70, 71).

The penultimate stage of myeloid development is the band granulocyte, which has an indented nucleus, but any ‘constriction’ in the width of the nucleus must be less than 50% of the total width. The nuclear membrane is smooth and chromatin is less densely clumped than in segmented cells. Secondary granules are present and the cytoplasm is less basophilic than previous stages of development. The ultimate stage of myeloid development is the mature, segmented granulocyte, the characteristics of which have been described in Chapter 4.

Monocytic cells

The developmental stages of monocytes include mono-blasts, promonocytes and monocytes. Monoblasts and promonocytes are morphologically very similar to some of the developmental stages of granulocytic cells (such as myeloblasts and promyelocytes). Monocytes in the bone marrow have a similar appearance to monocytes in the peripheral blood (as described in Chapter 4). Monocytes subsequently migrate from the blood, become resident in tissues (including the bone marrow) and differentiate into macrophages. Macrophages in the bone marrow typically have an ovoid to irregularly shaped nucleus composed of fine to reticular chromatin and a moderate to large amount of grey-basophilic cytoplasm, which may contain a granular, green-black pigment that represents haemosiderin. In some samples these iron-containing macrophages, also known as siderophages or ‘nurse cells’, may be observed in the centre of erythroblastic islands where iron is being distributed to erythroid precursor cells (Plate 68).

Lymphocytic cells

As previously mentioned, lymphopoiesis occurs in the bone marrow as well as in other tissues. The cells of lymphoid origin that may be recognised in bone marrow include lymphoblasts, prolymphocytes, mature lymphocytes and plasma cells. The least mature recognisable stage of lymphoid development is the lymphoblast. These are cells with a large nucleus, coarse chromatin, typically a single prominent nucleolus, a high nuclear to cytoplasmic ratio, and a small to moderate amount of basophilic cytoplasm. Prolymphocytes lack a nucleolus and compared with lymphocytes are larger (approximately the same size as a neutrophil), with less dense chromatin than the mature cells. Lymphoblasts and prolymphocytes may be difficult to distinguish from the similar stages of erythroid development (i.e. rubriblast and prorubricyte), particularly in histological samples.

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