CHAPTER ONE
Hematopoiesis

General Features

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 extramedullary 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 references in Bibliography section. 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 1.1 Overview of hematopoiesis.

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% hematopoietic cells and 50% 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 µm in diameter compared with approximately 20-30 µm for the largest granulocytic or erythrocytic precursors.

Figure 1.2 Histological section of canine bone marrow. Pink bony trabeculae are present in the lower left corner, lower right corner, and top of the photomicrograph and surround the hematopoietic cells and fat. The round to oval clear areas are the fat. The erythrocytic and granulocytic precursor cells are the many small, round purple structures. The larger, densely staining purple structures distributed throughout the marrow space are megakaryocytes. Canine bone marrow core biopsy; hematoxylin and eosin stain; 10× objective.

Figure 1.3 Megakaryocyte, erythrocytic precursors, and granulocytic precursors. The megakaryocyte is the largest cell located in right center of the field. The early erythrocytic precursors have central round nuclei and deep blue cytoplasm. The early granulocytic precursors have oval to indented nuclei and blue cytoplasm. There is a granulocytic predominance in this field. Canine bone marrow smear; 50× objective.

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 (Figures 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.

Figure 1.4 Erythrocytic precursors. The majority of the intact cells present are early erythrocytic precursors with centrally located round nuclei and deep blue cytoplasm. The cells with round eccentrically placed nuclei and reddish blue cytoplasm are late-stage erythrocytic precursors. The largest cell in the right center of the field that has small pink granules in the cytoplasm is a promyelocyte. Canine bone marrow smear; 100× objective.

Figure 1.5 Granulocytic precursors. The majority of the intact cells present are granulocytic precursors with oval to indented nuclei and blue cytoplasm. The larger immature forms have small, pink cytoplasmic granules. The cytoplasm becomes less blue as the cells mature. Canine bone marrow smear; 100× objective.

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.

Erythropoiesis

There are several stages of erythrocyte development that are recognizable in the bone marrow. Figure 1.6 depicts erythrocyte development, and Plate 1.1 shows the morphology of all erythrocytic precursors. Briefly, erythrocyte development is as follows.

Figure 1.6 Overview of erythropoiesis.

Rubriblast

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

Prorubricyte

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

Rubricyte

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

Metarubricyte

The metarubricyte is smaller than the rubricyte. The nucleus is round to oval, usually slightly eccentrically located, and has very condensed chromatin. There are small to moderate amounts of blue to reddish-blue cytoplasm. The metarubricytes, with more-reddish cytoplasm, contain more hemoglobin

Polychromatophil

The polychromatophil does not have a nucleus, and cytoplasm is blue to reddish-blue. As polychromatophils mature, they become less blue and more red as a result of their increased amounts of hemoglobin

Red blood cell

The red blood cell does not have a nucleus, and the cytoplasm is reddish to reddish-orange. The central pallor present here is a result of the biconcave discoid shape of the cells

Figure 1.15 Red blood cell development

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 z1. 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...