All blood cells have a finite life span, but in normal animals, the number of cells in circulation is maintained at a fairly constant level. To accomplish this, cells in circulation need to be constantly replenished, which occurs via the production and release of cells from the bone marrow. Production sites in the bone marrow are commonly referred to as medullary sites. In times of increased demand, production can also occur outside the bone marrow in sites such as spleen, liver, and lymph nodes. These sites are called extra-medullary sites. In rodents, in the normal steady state, extramedullary production of blood cells occurs in the spleen.
Hematopoiesis, the production of blood cells, is a complex and highly regulated process. Some differences in hematopoiesis exist between species and are beyond the scope of this text; readers are referred to the detailed coverage in some of the Selected References. The dog will be used to demonstrate some of the basic principles of hematopoiesis. All blood cells in the bone marrow arise from a common stem cell. This pluripotent stem cell gives rise to several stages of committed progenitor cells, which then differentiate into cells of the erythrocytic, granulocytic, megakaryocytic, and agranulocytic (monocytic and lymphocytic) lineages. The end result of this development process is the release of red blood cells, white blood cells, and platelets into the circulation. At the light microscopic level, without the use of immunocytochemistry or enzyme cytochemistry, it is impossible to accurately identify the early stem cells in the bone marrow, but the more differentiated stages of development can be identified and are graphically depicted in Figure 1.1.
Figure 1.2 shows a histological section of a bone marrow core biopsy from an adult dog. Note that there is a mixture of approximately 50 percent hematopoietic cells and 50 percent fat that is surrounded by bony trabeculae. The specific types of bone marrow cells can be difficult to recognize in histological sections at this low-power magnification, but the very large cells present are megakaryocytes. Cells are easier to identify on a smear from a bone marrow aspirate (Figure 1.3). The cells that are present include erythrocytic and granulocytic precursors and a megakaryocyte. To classify these three different cell types, there are some general features that can be used. Megakaryocytes are easy to distinguish by their very large size; the majority of them are 100-200 ?m in diameter compared with approximately 20-30 ?m for the largest granulocytic or erythrocytic precursors.
Cells of the erythrocytic lineage can be initially distinguished from those of the granulocytic lineage on the basis of their nuclear shape and color of cytoplasm (Figs. 1.4 and 1.5). Cells of the erythrocytic lineage have very round nuclei throughout most stages of development. In contrast, the nuclei of cells of the granulocytic lineage become indented and segmented as they mature. In addition, the cytoplasm of early erythrocytic precursors is much bluer than that of the granulocytic precursors.
There are several additional common morphological features that occur during development of both erythrocytic and granulocytic precursors. Both cell and nucleus decrease in size as they mature. As cells lose their capacity to divide, there is a loss of nucleoli and a condensation of nuclear chromatin. Changes in the cytoplasm are also occurring. As the hemoglobin content in erythrocytic precursors increases, the cytoplasm becomes less blue and more red. As maturation proceeds in the granulocytic cells, the cytoplasm also becomes less blue.
There are several stages of erythrocyte development that are recognizable in the bone marrow. Figure 1.6 depicts erythrocyte development, and Plate 1 (p. 10) shows the morphology of all erythrocytic precursors. Briefly, erythrocyte development is as follows.
The rubriblast is the first morphologically recognizable erythrocytic precursor. The rubriblast is a large, round cell with a large, round nucleus with coarsely granular chromatin and a prominent nucleolus. These cells have small amounts of deep blue cytoplasm. The rubriblast divides to produce two prorubricytes.
The prorubricyte is round and is of equal size or is sometimes larger than the rubriblast. The nucleus is round, with a coarsely granular chromatin pattern. A nucleolus is typically not present. There is a small amount of deep blue cytoplasm, often with a prominent perinuclear clear zone. Each prorubricyte divides to form two rubricytes.
The rubricyte is smaller than the prorubricyte. The nucleus is still round, and the coarsely granular chromatin is more condensed compared with the earlier stages. There is a small amount of deep blue cytoplasm, although some of the more mature rubricytes have reddish-blue cytoplasm. At the rubricyte stage, there are two divisions; the rubricytes then mature into metarubricytes.
The metarubricyte is smaller than the rubricyte. The nucleus is round to slightly oval, is centrally to eccentrically located, and has very condensed chromatin. There is a moderate amount of blue to reddish-blue cytoplasm. From the metarubricyte stage on, there is no further division of the cells, just maturation.
The highly condensed pyknotic nucleus of the metarubricyte is extruded from the cell, and this cell becomes a polychromatophil. Polychromatophils are round cells without a nucleus and have bluish cytoplasm. As a polychromatophil matures, it becomes less blue and more red, becoming a mature red blood cell. The mature red blood cells have species-dependent morphological features, which are described in Chapter 2.
Granulopoiesis is depicted in Figure 1.7 and Plate 2 (p. 11). In the bone marrow, there are three types of granulocytes, which include cells of the neutrophilic, eosinophilic, and basophilic lineages. Cells of the neutrophilic lineage are the predominant type of granulocyte present, and their development is described first.
The myeloblast is the first recognizable granulocytic precursor in the bone marrow. It is a large cell with a round to oval nucleus with a finely granular chromatin pattern and one or more prominent nucleoli. The amount of cytoplasm is small to moderate and blue. Each myeloblast divides to form two promyelocytes. Promyelocytes look similar to myeloblasts except they may not have nucleoli, and they may have a perinuclear clear zone within the cytoplasm. The distinguishing feature of promyelocytes is that they contain multiple, very small, pink to purple granules in the cytoplasm; these are known as primary granules. Promyelocytes divide to produce myelocytes.
The myelocyte is smaller than the earlier precursors and has a round to oval to slightly indented nucleus with finely to moderately granular chromatin. These cells have moderate amounts of blue cytoplasm. At this stage, primary granules are no longer being produced, and now secondary granules are formed. These secondary granules are larger than the primary granules.
In neutrophilic myelocytes, the secondary granules are light pink and are very difficult to recognize with the light microscope. The myelocyte goes through two divisions, and the resulting progeny mature into metamyelocytes. From the metamyelocyte stage forward, the cells no longer divide.
The metamyelocyte is smaller than the myelocyte and has a kidney-shaped nucleus. The chromatin is moderately granular and is more condensed and clumped than that in the myelocyte. The cytoplasm is blue and contains primary and secondary granules. Both types of granules in the metamyelocyte and subsequent stages of development are not easily seen light microscopically in most animal species. Metamyelocytes develop into band neutrophils. Band neutrophils are round and smaller than metamyelocytes, have horseshoe-shaped nuclei, and have moderate amounts of blue to light blue cytoplasm. The band neutrophil will mature into a segmented neutrophil, which is a small cell with faintly blue to pink cytoplasm and a segmented nucleus. The nuclear chromatin is coarsely granular and clumped.
Mature eosinophils and basophils and their precursors are found in very low numbers in the normal bone marrow. The production of these cells is very similar to that of neutrophils, and the only major differences are described in Figure 1.1. The development is identical until the myelocytic stage, which is when eosinophilic and basophilic myelocytes can be distinguished from neutrophilic myelocytes by the color of the secondary granules. The eosinophilic and basophilic myelocytes contain reddish to reddish-orange and purple secondary granules, respectively. Eosinophilic and basophilic metamyelocytes and bands can also be recognized by the presence of the unique secondary granules.
The last stage of development is the mature eosinophil and basophil. The eosinophil is often slightly larger than the mature neutrophil, and the nucleus is not as tightly segmented. The cytoplasm contains reddish to reddish-orange granules. The mature basophil is a round cell that is slightly larger than the neutrophil, with a segmented nucleus with condensed chromatin. The cytoplasm is light purple and may contain granules. There are some unique species-dependent features of mature eosinophils and basophils, which are described in Chapter 5.
The precursors of monocytes arise from committed stem cells, which are common precursors for both cells of the granulocytic and monocytic lineage. Monocyte development is depicted in Figure 1.1. In normal bone marrow, very few cells of the monocytic lineage are present. Monoblasts are the first microscopically recognizable precursors in bone marrow, although they can be impossible to differentiate from myeloblasts. Monoblasts give rise to promonocytes. A promonocyte is a large cell with an oval to sometimes indented nucleus with a reticular (netlike) or lacy chromatin pattern. These cells have small to moderate amounts of blue cytoplasm and can be difficult to distinguish from neutrophilic myelocytes or metamyelocytes. Promonocytes give rise to monocytes, which are larger than segmented neutrophils. The nucleus of the monocyte has multiple indentations. The nuclear chromatin has areas of condensation but has a lacy or reticular pattern compared with the condensed chromatin pattern of the mature neutrophil. The cytoplasm is moderate in amount and is typically blue-gray, often with discrete multiple vacuoles.
Megakaryocytopoiesis and Platelet Production
Megakaryocytopoiesis is quite unique compared with the development of the other blood cells and is depicted in Figure 1.1. The megakaryoblasts are the first morphologically recognizable precursors of the megakaryocytic lineage in bone marrow but can be impossible to differentiate from other blast cells. The megakaryoblast is a large cell with a single round nucleus and prominent nucleolus. This cell differentiates into a promegakaryocyte, which is larger than the megakaryoblast and has a multilobed nucleus with dark blue agranular cytoplasm. The promegakaryocyte gives rise to the megakaryocyte (Figure 1.8), which is easily recognized in the bone marrow because of its large size (typically 100-200 ?m). This large cell has a large, multilobulated nucleus and abundant granular cytoplasm.
Platelets are formed from the cytoplasm of megakaryocytes by the formation of a structure known as a proplatelet. The proplatelet is fragmented into multiple platelets. The resulting platelets are discoid-shaped small cells that do not have nuclei and that have light pink cytoplasm with sometimes distinct purple granules.
Lymphocytes arise from the same common stem cell precursor as the other bone marrow cells (Fig. 1.1). Multiple stages of differentiation of lymphocytes in bone marrow cannot be recognized light microscopically, but there are two main types of lymphocytes that can be identified by immunophenotyping in the peripheral blood: B and T lymphocytes. These two cell types look similar and cannot be differentiated on the basis of morphology alone, but their functions are quite different. In bone marrow, low numbers of small lymphocytes and rarely seen medium and large lymphocytes are present (Fig. 1.9). The exact number of lymphocytes present in bone marrow is species dependent; however, rodents have a relatively greater abundance of bone marrow lymphocytes compared with the common domestic species.
The small lymphocyte is a small, round cell with a round to slightly indented nucleus. In some areas, the nuclear chromatin has a very smooth glassy appearance, and in other areas it is more clumped or smudged. Overall, the chromatin is not as condensed as that of a rubricyte, which is the cell type with which it is most often confused. The lymphocyte has a small amount of light blue cytoplasm. The medium and large lymphocytes, as the names imply, are larger than the small lymphocytes. The nuclei are round, and the chromatin is finely granular, with some areas of condensation. The nucleus of the large lymphocyte typically has a nucleolus and is known as a lymphoblast. Both cell types have small amounts of light to moderate blue cytoplasm.
In addition to lymphocytes, low numbers of plasma cells can be seen in bone marrow (Fig. 1.10). These cells are the end stage of differentiation of B lymphocytes and are round with eccentrically placed round nuclei. The nuclear chromatin is very condensed and clumped, with clear areas between the clumps. Plasma cells have moderate amounts of deep blue cytoplasm with a prominent perinuclear clear zone.
Other Cells of the Bone Marrow
Bone marrow macrophages are present in low numbers (Fig. 1.11). These cells are large and have an oval to indented nucleus. The nuclear chromatin is reticular (netlike). The moderate to abundant amounts of blue cytoplasm often are very foamy and may contain multiple, variably sized vacuoles. Often within these cells there can be phagocytized debris or iron pigment, known as hemosiderin. In general, hemosiderin is not identified in normal cat bone marrow but is readily identifiable in the marrow of most other common domestic and non-domestic species.
Osteoclasts are rarely found in bone marrow smears. Osteoclasts are similar in size to megakaryocytes, and these two cell types are often confused (Fig. 1.12). The osteoclast has multiple, individual, round to oval nuclei. In contrast, the nucleus of the mature mega-karyocyte is multilobulated. The osteoclast cytoplasm is granular and light blue to red.
Osteoblasts are also found in very low numbers in normal bone marrow. The size of these cells is very similar to that of the macrophage, and the morphology is somewhat similar to that of the plasma cell, including an eccentrically placed round nucleus and prominent perinuclear clear zone (Fig. 1.13). The nucleus has a granular chromatin pattern, usually with a prominent single nucleolus. The cytoplasm is a light to moderate blue. In contrast, the plasma cell is smaller in size, and the nuclear chromatin is much more condensed, with no prominent nucleoli.
PLATE 1. Red Blood Cell Development
The rubriblast is a large, round cell with a large, round nucleus; coarsely granular chromatin; and a nucleolus. This cell has small amounts of deep blue cytoplasm.
The prorubricyte is a large, round cell with a round nucleus with a coarsely granular chromatin pattern. This cell typically lacks a nucleolus. There is a small amount of deep blue cytoplasm often with a prominent perinuclear clear zone.
The rubricyte is a round cell with a round, centrally located nucleus; it is smaller than the prorubricyte. The coarsely granular chromatin is more condensed compared with the earlier stages of development, and irregular clear areas are present between the chromatin clumps. The cytoplasm varies from deep blue to reddish-blue. Early rubricytes typically have more bluish cytoplasm, and later rubricytes stain more red as the amount of hemoglobin increases.