I. General Features of Hematopoiesis
Hematopoiesis is blood cell production. It involves the proliferation and differentiation of hematopoietic stem cells and may be subdivided, according to the cell type formed, into erythropoiesis, leukopoiesis, granulopoiesis, agranulopoiesis, lymphopoiesis, and thrombopoiesis.
A. Hematopoietic Stem Cells
These are undifferentiated mesodermal derivatives able to divide repeatedly and differentiate into mature blood cells. The nature and structure of the earliest blood cell precursors are debatable. The best available evidence supports the monophyletic theory of hematopoiesis, according to which a single pluripotent stem cell can form all mature blood cell types. Hematopoietic stem cells are called colony-forming cells (CFCs), or colony-forming units (CFUs), because they form colonies of recognizable blood cell types in culture. Pluripotent CFCs were first demonstrated in spleen cell cultures and are called CFC-S cells. Some CFC-S cells may circulate in a form resembling lymphocytes. CFC-S cells divide only rarely, perhaps because each of their progeny can give rise to so many cells. The progeny of a dividing CFC-S cell remains pluripotent or differentiates into one of several unipotential stem cell types, which can divide but each of which produces only one mature blood cell type (e.g., CFC-E cells form erythrocytes).
These tissues are collections of CFCs and their progeny at various stages of maturation suspended in a reticular connective tissue stroma. Active hematopoiesis shifts its location in overlapping stages during development (II.A.1–3): It occurs first in the extraembryonic mesoderm of the yolk sac; next in the fetal liver, spleen, and thymus; and finally in the bone marrow and lymphoid tissue.
Bone marrow (medullary tissue, III.A) is the primary hematopoietic tissue from the fifth month of fetal life. All bone marrow begins as active, or red, marrow. All bone marrow contains abundant adipocytes and a reticular connective tissue stroma. During growth, development, and aging, portions of the red marrow are replaced by increasing numbers of adipocytes to form yellow marrow. Yellow marrow can be reactivated by an increased demand for blood cells (e.g., during chronic hypoxia and hemorrhage). Yellow marrow does not produce blood cells and thus is not a hematopoietic tissue. Red marrow has a limited distribution in adults. It contains masses of reticular connective tissue stroma that support the CFCs and their progeny (the hematopoietic cords), separated by vascular sinusoids whose walls have openings through which maturing blood cells enter the circulation.
Blood cells have a limited life span in the circulation, owing to the recognition and removal of worn and damaged erythrocytes by macrophages and to the migration of leukocytes into the surrounding tissues. To keep constant numbers of each cell type in circulation, hematopoiesis must be continuous. Otherwise, a decrease in the number of circulating cells, or anemia, results.
E. Regulation of Hematopoiesis
Regulation involves colony-stimulating factors (CSFs), such as erythropoietin, leukopoietin, and thrombopoietin. These hormones act at various steps in hematopoiesis to enhance the proliferation and differentiation of CFCs. Erythropoietin (VII.A) stimulates erythropoiesis. The discovery of a variety of CSFs (e.g., GM-CSF, G-CSF, M-CSF, and steel factor) with overlapping hematopoietic activities has provided a basis for therapeutic management of conditions that would otherwise result in leukopenia.
II. Development of Hematopoietic Tissues
A. Sites of Intrauterine Hematopoiesis
Primordial (prehepatic) phase. During week 3 of embryonic development, cell clusters called blood islands form in the extraembryonic mesoderm of the yolk sac. Cells at the periphery form the endothelium of the primitive blood vessels. By a process called megaloblastic erythropoiesis, cells at the center form the first blood cells, called primitive erythroblasts. These differ from definitive erythroblasts of later stages in that they are larger, contain a unique type of hemoglobin, and retain their nuclei. Leukocytes and platelets do not appear until the next phase.
Hepatosplenothymic phase. During the second month, hematopoiesis shifts to the liver, spleen, and thymus. Hematopoietic stem cells invade these organs and begin producing a wider variety of blood cell types. The liver produces granulocytes, platelets, and red blood cells that may be nucleated (definitive erythroblasts) or enucleate (erythrocytes). Hematopoiesis in the liver declines during the fifth month, but continues at low levels until a few weeks after birth. The spleen produces mainly erythrocytes and small numbers of granulocytes and platelets. Just before birth, lymphopoiesis becomes an important splenic function. The thymus produces T lymphocytes, which assume a variety of specialized functions (14.III.A.2).
Medullolymphatic (definitive) phase. During the third month, hematopoiesis begins shifting to the bone marrow and lymphoid tissue, where it remains throughout adulthood. Medullary tissue (bone marrow) first becomes hematopoietic in the clavicle's diaphysis, between months 2 and 3. As other bones ossify, their marrow becomes active. By the fifth month, bone marrow is the primary hematopoietic tissue, producing platelets and all blood cell types. Additional lymphocytes form in the developing lymphoid tissues and organs (e.g., thymus, lymph nodes, spleen). Before birth, the lymph nodes also may produce red blood cells.
B. Sites of Postnatal Hematopoiesis
Beginning in infancy, hematopoiesis is restricted to the bone marrow (medullary or myeloid tissue) and the lymphoid tissues.
C. Extramedullary Hematopoiesis in Disease
In adults, erythropoiesis, granulopoiesis, and thrombopoiesis in sites other than bone marrow are abnormal. When bone marrow cannot meet the demand for blood cells, the liver, spleen, or lymph nodes may resume their embryonic hematopoietic activity.
III. General Structure of Mature Hematopoietic Tissue
Mature hematopoietic tissues share a basic architecture supported by a reticular connective tissue scaffolding (stroma) permeated by many sinusoids. The meshwork between the sinusoids contains developing blood cells; as these complete their differentiation, they enter the circulation through openings in the sinusoid walls.
All marrow begins as red marrow, also called active, or hematogenous, marrow. During growth, the blood cells are gradually depleted and are replaced by adipocytes. In adults, red marrow is restricted to the skull, vertebrae, ribs, sternum, ilia, and the proximal epiphyses of some long bones. The fatty, nonhematopoietic replacement tissue in other bony cavities is termed yellow marrow.
Stroma consists of adipocytes (as much as 75% of red marrow), macrophages, and reticular connective tissue composed of reticular cells (adventitial cells) and the reticular fibers (type III collagen) they produce. Reticular cells are highly branched, mesenchymal derivatives resembling fibroblasts. Their processes separate the developing blood cells from the endothelium of sinusoids.
Hematopoietic cords, which comprise the stromal scaffolding, are crowded with overlapping blood cells of all types and at all stages of differentiation. Nests of similar cells, often the progeny of a single stem cell, occupy different microenvironments in the marrow cords. Abundant sinusoids lie between the cords and have openings in their walls through which maturing blood cells and platelets enter the circulation. In histologic section, the dense packing makes the identification of individual cell types difficult. Differentiating blood cells are therefore commonly studied in smears.
Bone marrow functions. In addition to being the primary site for hematopoiesis, bone marrow helps destroy old red blood cells. Macrophages in the bone marrow, spleen, and liver break down hemoglobin to form (1) globin, which is quickly hydrolyzed; (2) porphyrin rings, which are converted to bilirubin; and (3) iron, which is complexed with and transported by the plasma protein transferrin to other bone marrow sites for reuse by developing erythrocytes. Iron is stored in bone marrow macrophages as ferritin (iron complexed with the protein apoferritin) and hemosiderin. In some sections, clusters of developing erythrocytes surround and receive iron from macrophages in groupings called erythroblastic islands.
B. Lymphoid Tissues and Organs
The thymus, spleen, lymph nodes, and lymphatic aggregations, such as the tonsils and Peyer's patches, contribute to postnatal hematopoiesis by providing sites for lymphocyte proliferation, programming, and differentiation (lymphopoiesis). Lymphoid organs and tissues are also assembled on a reticular connective scaffolding and are described in Chapter 14.
In healthy adults, erythropoiesis (red blood cell formation) occurs exclusively in the bone marrow. Erythrocytes derive from CFC-E cells, which in turn derive from CFC-S cells. Erythrocyte differentiation is commonly described by naming cell types at specific stages in the process according to their histologic characteristics (IV.B). Cellular changes that occur during erythroid differentiation include (1) a decrease in cell size, (2) condensation of nuclear chromatin, (3) a decrease in nuclear diameter, (4) an accumulation of hemoglobin in the cytoplasm (increased acidophilia), (5) a decline in ribosome numbers in the cytoplasm (decreased basophilia), and (6) ejection of the nucleus.
B. Stages of Erythroid Differentiation
Erythrocyte maturation is commonly divided into six stages (Fig. 13–1). These stages are identified by overall cell diameter, nuclear size and chromatin pattern, and cytoplasmic staining properties. Cells in transition between these stages are commonly found in bone marrow smears. Cell division occurs throughout the early stages, but cells lose their ability to divide during the normoblast stage. The following discussion begins with the least mature cells; the sixth (final) stage produces the mature erythrocyte (12.III.A.1).
Proerythroblasts are large (14–19 μm in diameter) and contain a large, centrally located, pale-staining nucleus with one or two large nucleoli. The small amount of cytoplasm (approximately 20% of cell volume) contains polyribosomes actively synthesizing hemoglobin. The resulting cytoplasmic basophilia allows these cells to be distinguished from myeloblasts, with which they are most easily confused. Proerythroblasts are capable of multiple mitoses and may be considered unipotential stem cells.
Basophilic erythroblasts are slightly smaller than proerythroblasts, with a diameter of 13 to 16 μm. They have slightly smaller nuclei with patchy chromatin. Their nucleoli are difficult to distinguish. The cytoplasm is more intensely basophilic, typically staining a deep royal blue. A prominent, clear, juxtanuclear cytocenter is often visible. Basophilic erythroblasts continue hemoglobin synthesis at a high rate and are capable of mitosis.
Polychromatophilic erythroblasts are smaller yet (12–15 μm in diameter), and more hemoglobin accumulates in their cytoplasm. The conflicting staining affinities of the polyribosomes (basophilic) and hemoglobin (acidophilic) give the cytoplasm a grayish appearance. The nucleus is smaller than in less mature cells, with more condensed chromatin forming a checkerboard pattern. Cells at this stage retain the ability to synthesize hemoglobin and to divide.
Normoblasts (orthochromatophilic erythroblasts) are easily identified because of their small size (8–10 μm in diameter); an acidophilic cytoplasm with only traces of basophilia; and small, eccentric nuclei with chromatin so condensed that it appears black. Although early normoblasts may divide, erythroid cells lose their ability to do so during this stage, which ends with the extrusion of the pyknotic (degenerated, dead) nucleus.
Reticulocytes are nearly indistinguishable from mature erythrocytes with standard stains; however, when they are stained with the supravital dye cresyl blue, residual polyribosomes form a blue-staining, netlike precipitate in the cytoplasm. Reticulocytes complete their maturation to become erythrocytes (12.III.A.1) during their first 24 to 48 hours in circulation. This process involves the ejection or enzymatic digestion of their remaining organelles and assumption of the biconcave shape.
Erythropoiesis. Schematic diagram of erythrocyte precursor cells at various stages of erythroid development. The proerythroblast derives from a CFU-E cell. Drawings are roughly to scale.
Leukopoiesis (white blood cell formation) encompasses both granulopoiesis and agranulopoiesis. Leukopoietic CFCs that have been identified include CFC-GM (forms both granulocytes and macrophages), CFC-G (forms all granulocyte types), CFC-M (forms macrophages), and CFC-EO (forms only eosinophils). All of these CFCs with limited capabilities derive from the pluripotential CFC-S cells.
General. Granulopoiesis occurs in the bone marrow of healthy adults. The three granulocyte types—neutrophils, basophils, and eosinophils—may all derive from a single precursor (CFC-G). The structural changes that characterize granulopoiesis include (1) a decrease in cell size, (2) condensation of nuclear chromatin, (3) changes in nuclear shape (flattening → indentation → lobulation, a progression resembling the gradual deflation of a balloon), and (4) an accumulation of cytoplasmic granules.
Stages of granulocyte differentiation. Granulocyte maturation is commonly divided into six stages (Fig. 13–2). These stages are identified by overall cell diameter; size, shape, and chromatin pattern in the nuclei; and type and number of specific granules in the cytoplasm. The specific granules, with their characteristic staining properties, first appear at the myelocyte stage; from this point, the cells are named according to the mature granulocyte type they will form (e.g., neutrophilic myelocyte). In the granulocyte series, cell division ceases at the metamyelocyte stage. The following discussion begins with the least mature cells; the sixth (final) stage produces the mature granulocyte (12.III.B.2.a–c).
Myeloblasts, the earliest recognizable granulocyte precursors, are approximately 15 μm in diameter and are difficult to distinguish from other stem cells. Each has a large, spherical, euchromatic nucleus with as many as three smudgy nucleoli. Their cytoplasm lacks granules and is more basophilic than that of their CFC precursors but less basophilic than that of proerythroblasts, with which they are most often confused.
Promyelocytes (15–24 μm in diameter) are larger than myeloblasts and their chromatin is slightly more condensed. Their otherwise spherical nuclei may be flattened on one side and may contain nucleoli. Their cytoplasm is more basophilic than that of myeloblasts and contains azurophilic granules (0.05–0.25 μm in diameter) but not specific granules (12.III.B), which appear during the subsequent stage. Because azurophilic granules are synthesized mainly during this stage, the number per cell decreases during subsequent division and maturation. These granules contain lytic enzymes and function as lysosomes.
Myelocytes typically are smaller than promyelocytes (10–16 μm in diameter). This is the first stage at which enough specific granules accumulate in the cytoplasm to enable distinction among the three immature granulocyte types: neutrophilic myelocytes, eosinophilic myelocytes, and basophilic myelocytes. Myelocyte nuclei are round to kidney-shaped, with chromatin that is more condensed than during previous stages. Like their precursors, myelocytes can divide.
Metamyelocytes. The three metamyelocyte types—neutrophilic, eosinophilic, and basophilic—are smaller (10–12 μm in diameter) and more densely packed with specific granules. The nucleus is deeply indented, often resembling a mask, and its chromatin is more condensed. During this stage, the capacity for mitosis is lost.
Band cells. The three band cell types—neutrophilic, eosinophilic, and basophilic—have horseshoe-shaped nuclei. They range in diameter from 10 to 12 μm. Like the erythroid reticulocytes, these nearly mature cells circulate in small numbers (3%–5% of circulating leukocytes) but may appear in larger numbers (commonly called a “shift to the left”) when granulopoiesis is hyperstimulated. During final maturation, the nuclei undergo further chromatin condensation and lobulation. Mature granulocytes (i.e., neutrophils, eosinophils, and basophils; Chapter 12) also occur in bone marrow.
Agranulocytes (monocytes and lymphocytes), like the other blood cell types, derive from CFC-S cells. The morphologic changes during maturation include decreases in overall cell and nuclear diameter and an increase in nuclear chromatin density. The morphologic characteristics of agranulocytes at immature stages are much less distinct than those of erythrocytes and granulocytes.
Monocytopoiesis. CFC derivatives that give rise to monocytes are called monoblasts and are difficult to identify in bone marrow smears. Monoblast derivatives, promonocytes, are slightly easier to identify and serve as immediate precursors of monocytes. Promonocytes are larger (10–20 μm in diameter) than monocytes and have pale-staining nuclei and basophilic cytoplasm.
Lymphopoiesis. In adults, lymphopoiesis occurs mainly in lymphoid tissues and organs and to a lesser extent in bone marrow. Before division, the precursor, or lymphoblast, is larger than the typical circulating lymphocyte. However, many circulating lymphocytes can respond to antigenic stimulation by blasting (enlarging to assume lymphoblast morphology) and then proceeding to divide. Some of these, called null cells, are neither T nor B cells and may represent circulating CFC-S cells.
Granulopoiesis. Schematic diagram of granulocyte precursor cells at various stages of granulocyte development. Drawings are roughly to scale.
Platelet (thrombocyte) production is carried out in the bone marrow by unusually large cells (100 μm in diameter) called megakaryocytes. Immature megakaryocytes, called megakaryoblasts, derive from CFC-Megs, which in turn derive from CFC-S cells. Megakaryoblasts undergo successive incomplete mitoses involving repeated DNA replications without cellular or nuclear division. The result of this process, called endomitosis, is a single large megakaryocyte with a single, large, multilobed, polyploid (as many as 64 n) nucleus. Maturation involves lobulation of the nucleus and development of an elaborate demarcation membrane system that subdivides the peripheral cytoplasm, outlining cytoplasmic fragments destined to become platelets. As the demarcation membranes fuse to form the plasma membranes of the platelets, ribbonlike groups of platelets are shed from the megakaryocyte periphery into the marrow sinusoids to enter the circulation.
VII. Compartments & the Life Cycle of Blood Cell Types
The total population of mature and developing red blood cells constitutes the widely dispersed but functionally discrete erythron, which is subdivided into two compartments. The circulating compartment includes all mature erythrocytes in the circulation (approximately 2.5 × 1013). The medullary compartment (erythropoietic pool) includes the bone marrow sites where erythropoiesis occurs. Erythrocytes usually leave the bone marrow to enter the circulation as reticulocytes and undergo final maturation within 24 to 48 hours. Mature erythrocytes circulate for approximately 120 days before they are retired by macrophages (primarily in the spleen, but also in the bone marrow and liver). Approximately 1011 erythrocytes are retired daily. The iron in the hemoglobin is conserved and eventually returned to the marrow by transferrin. Iron-free hemoglobin is converted by the liver into bile pigment called bilirubin. Red cell replacement is controlled by the glycoprotein hormone erythropoietin, which stimulates erythrocyte precursors in the bone marrow to proliferate and differentiate. Erythropoietin is produced by fibroblastlike cells in the kidney cortex in response to low oxygen tension in the blood. Other factors affecting erythrocyte production and function include iron, intrinsic factor, vitamin B12, and folic acid.
Neutrophils and other granulocytes are continually produced in the bone marrow and, because their numbers remain relatively constant, they also must be continually destroyed. Granulocytes constantly move from the marrow to the circulation to the tissues, where most of them die.
The medullary formation compartment in the bone marrow comprises the stem cells and is the site of granulopoiesis. Cells spend approximately 7 days in this compartment.
The medullary reserve compartment in the bone marrow comprises newly formed granulocytes that have yet to enter the circulation. Neutrophils remain here for another 4 days.
The circulating compartment comprises mature granulocytes circulating in the blood. The number of cells in the circulating compartment remains relatively constant, even though most granulocytes circulate for only a few hours. When the cell number in this compartment decreases as a result of margination or removal of the cells from the blood (e.g., by leukopheresis), granulocyte production in the bone marrow is stimulated to replace the missing cells by multiple CSFs (I.E.), which together are called leukopoietins.
The marginating compartment comprises cells that have entered the circulation but have attached to the walls of blood vessels, become confined by vasoconstriction in some capillary beds, or passed through intercellular junctions between endothelial cells to move out of the blood vessels and into the connective tissues—a process called diapedesis. After they have entered the tissues, granulocytes rarely reenter the circulation. However, exchanges between the rest of the marginating compartment and the circulating compartment occur continuously. The total time spent in the circulating and marginating compartments is approximately 6 to 7 hours.
Precursors of both B cells and T cells are produced in the bone marrow. Those destined to become T cells migrate to the thymus, where they are programmed to assume the specialized functions of this lymphocyte class before reentering the circulation and moving to the spleen or lymph nodes for final maturation. Mature T cells return to the circulation for a long period of time; in humans, they have a life span that is measured in years. Precursors destined to become B cells never enter the thymus but are programmed as B cells in the bone marrow and are subsequently distributed to the spleen, lymph nodes, and other lymphatic aggregations, where they respond to specific antigens. B cells have a life span of at least 6 weeks in humans. In response to antigenic stimulation, they proliferate and differentiate into plasma cells. Lymphopoiesis and lymphocyte function are discussed further in Chapter 14.
Monocytes form in the bone marrow and remain in circulation for approximately 2 days before passing between the endothelial cells in the walls of capillaries and venules. They enter the connective tissues to differentiate into macrophages and other mature components of the mononuclear phagocyte system, including the Kupffer cells in the liver and osteoclasts in bone.
Platelets are formed in the bone marrow, most likely in response to increased blood levels of one or more CSFs referred to as thrombopoietin. Platelets have a life span of approximately 10 days in the circulation. Aside from their involvement in clot formation and the eventual removal of clots by sloughing or phagocytosis, the fate of platelets is unclear.