Histology for Pathologists

Editors: Mills, Stacey E.

Title: Histology for Pathologists, 3rd Edition

Copyright 2007 Lippincott Williams & Wilkins

> Table of Contents > VIII - Hematopoietic System > 33 - Bone Marrow

33

Bone Marrow

S. N. Wickramasinghe

Introduction

The bone marrow is a large and complex organ that is distributed throughout the cavities of the skeleton. The total mass of the bone marrow of an adult has been estimated to be 1600 to 3700 g, exceeding that of the liver. About half this mass consists of hematopoietically inactive fatty marrow (which appears yellow) and the remainder of hematopoietically active marrow (which appears red). Although essentially hematopoietically inactive, even fatty marrow contains a few scattered microscopic foci of hematopoietic cells. The functions of hematopoietic marrow include: (a) the formation and release of various types of blood cells (hematopoiesis), mast cells, osteoclasts and some endothelial progenitor cells; (b) the phagocytosis and degradation of circulating particulate material such as microorganisms and abnormal or senescent red cells and leukocytes; and (c) antibody production. Recent studies indicate that, in addition to hematopoietic stem cells, the marrow contains mesenchymal stem cells that can differentiate under appropriate conditions into adipocytes, hepatocytes, osteoblasts and osteocytes, chondrocytes, skeletal and cardiac muscle cells, kidney cells and neural cell lineages, but the interpretation of some of the data is still being debated (1). The nonhematopoietic marrow serves as a large store of reserve lipids. The various functions of hematopoietic marrow are based on a high degree of structural

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Techniques for Studying the Marrow

The microscopic structure of the human bone marrow can be studied during life by performing a trephine biopsy of the posterior superior iliac spine or anterior iliac crest. This provides a core of bone and associated marrow. The biopsy specimen is commonly fixed in 10% neutral buffered formalin for 6 to 18 hr (depending on its width) but may be fixed in Zenker's solution for a minimum of 4 hr or in B5 (formalin and mercury chloride) for 4 hr. The fixed specimen is decalcified in 10% formic acid and 1% formalin or in one of a number of other decalcifying reagents. It is then processed in the usual manner and embedded in paraffin (2,3,4). Decalcification and paraffin embedding result in some shrinkage of marrow tissue, loss of the activity of cellular enzymes, and, sometimes, blurring of nuclear staining. In addition, certain decalcification procedures cause leaching of the iron stores (i.e., of the hemosiderin present within macrophages). However, the reactivity of some antigens with antibody is retained.

Histologic studies also can be performed on aspirated marrow. Two methods are in use. The first is to allow the marrow to clot before fixation and subsequent processing to paraffin. The clot sections obtained usually show only a few marrow fragments within a large mass of clotted blood. The second approach is to concentrate the marrow fragments by filtration or some other procedure before processing. This approach yields better preparations than do clot sections.

Sections of paraffin-embedded marrow fragments or decalcified bone cores are cut to a thickness of 3 to 5 m and are routinely stained with hematoxylin and eosin (H&E), with the Giemsa's stain, by a silver impregnation technique for reticulin, and by Perls' acid ferrocyanide method for hemosiderin (Prussian blue reaction). They may also be stained by the periodic acid-Schiff (PAS) reaction for glycogen or glycoprotein. A Leder's stain for chloroacetate esterase may be performed on sections of marrow fragments but works poorly on decalcified specimens (2). A limited range of antibodies can be used for immunohistochemical studies on formalin- or B5-fixed, paraffin-embedded sections of trephine biopsy specimens, using an immunoalkaline phosphatase or immunoperoxidase method. The most useful antibodies and the cell types they recognize are given in Table 33.1 (5,6,7,8,9,10,11).

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The cores obtained by trephine biopsy also may be fixed in a mixture containing formaldehyde, methanol, and glucose phosphate buffer and embedded in methyl methacrylate without decalcification (4,12,13,14,15). Semithin sections (1 3 m thick) of the undecalcified methyl methacrylate embedded material then may be cut using a special heavy-duty microtome. Such sections show cellular features in much greater detail than do paraffin-embedded sections but have no antigenic or enzymic reactivity. On the other hand, if the core is appropriately fixed and embedded in glycol methacrylate (a water-miscible plastic) or a mixture of methyl and glycol methacrylate, a number of antigen epitopes and enzyme activities are preserved (16,17,18).

Methyl methacrylate embedded semithin sections may be stained (after removing the methacrylate) with H&E or gallamine blue Giemsa for cellular detail, Gomori's stain for reticulin fibers, PAS stain, Berlin blue stain for iron, and Ladewig's and Goldner's stains for osteoid, calcified bone, and connective tissue (14,15,19,20,21). Trephine biopsy cores that are embedded in a mixture of methyl methacrylate and glycol methacrylate may be used for the demonstration of chloroacetate esterase, acid phosphatase, peroxidase, nonspecific esterase, and alkaline phosphatase, as well as for the immunohistochemical detection of some antigens (16,17).

For immunohistochemical studies with the widest range of antibodies, frozen sections of trephine biopsy cores must be used (22,23). However, such sections show less cytologic detail than paraffin-embedded sections and tend to become distorted, making interpretation difficult. The distortion when cutting frozen sections can be reduced using a special supporting medium such as Histocon (Polysciences; Warrington, Pennsylvania), which does not impair antigenic reactivity.

The trephine biopsy core can be used to prepare several imprints by gently touching it with glass slides before fixation for histology. The imprints so obtained are used for detailed investigations into the morphology and other characteristics of individual marrow cells. However, such studies are best performed on smears of aspirated marrow. In adults, marrow is aspirated from the posterior superior iliac spine or the iliac crest. In children, marrow is usually aspirated from the posterior superior iliac spine and, in the case of patients less than 1 year of age, also from the upper end of the medial surface of the tibia just below and medial to the tibial tuberosity. The imprints from the trephine biopsy core and marrow smears are usually stained by a Romanowsky method such as the May-Gr nwald-Giemsa (MGG) stain and also by Perls' acid ferrocyanide method. The smears also may be briefly fixed under conditions that preserve enzyme activity and antigenic sites and used to perform cytochemical and immunocytochemical studies. The details of these techniques and their value in hematologic diagnosis have been discussed by Hayhoe and Quaglino (24). Antibodies useful in immunocytochemical studies of appropriately fixed smears include those listed in Table 33.1, as well as several others such as those against CD2 (a pan-T marker), CD19 (a pan-B marker), and CD41 or CD42b (megakaryocyte lineage markers).

A thorough study of the marrow requires examination of both marrow smears and tissue sections. Marrow smears are the best preparations for the study of cellular detail but provide little information on intercellular relationships and the organization of the marrow. Histologic sections provide this information and are therefore superior to marrow smears for the detection of tumor infiltration, granulomas, amyloidosis, and necrosis of the marrow. They are essential for studying the distribution and quantity of extracellular reticulin and collagen fibers and may show vascular lesions (e.g., in thrombotic thrombocytopenic purpura and polyarteritis nodosa).

When electron microscopic studies are to be performed, an aliquot of a marrow aspirate is mixed with heparinized Hanks' solution. A few marrow fragments are then removed without delay and placed in a solution of 2.5 to 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). Alternatively, 1-mm pieces of the trephine biopsy core are fixed in glutaraldehyde for 1 hr, after which the marrow is gently teased out of the bone using a dissecting microscope.

In this chapter, unless otherwise stated, the descriptions of cells in marrow smears apply to smears stained by a Romanowsky method. The electron microscopic data relate to ultrathin sections stained with uranyl acetate and lead citrate. Such sections were prepared from marrow fragments that were fixed in glutaraldehyde and postfixed in osmium tetroxide.

General Features of Hematopoiesis

Blood cells are produced in the embryo and fetus and throughout postnatal life. In the developing fetus and growing child, the total number of hematopoietic cells and blood cells increases progressively with time. By contrast, the hematopoietic systems of healthy adults are examples of steady-state cell renewal systems. In such systems, a relatively constant rate of loss of mature blood cells from the circulation is balanced by the production of new blood cells at the same rate. The number of hematopoietic cells and blood cells therefore remains constant.

New blood cells are eventually derived from a small number of hematopoietic stem cells (25), which are estimated to constitute 1 in 10⁴ nucleated marrow cells. These cells have two properties: (a) the ability to mature into all types of blood cell and (b) an extensive capacity to

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Small numbers of bone marrow-derived hematopoietic stem cells and early hematopoietic progenitor cells circulate in the blood and these can home to the marrow.

Two processes are involved in the formation of all types of blood cells. These are the progressive acquisition of the biochemical, functional, and morphologic characteristics of the particular cell type (i.e., differentiation) and cell proliferation. The latter results in the production of a large number of mature cells from a single cell committed to one or more differentiation pathways. Differentiation occurs at all stages of hematopoiesis, and cell proliferation occurs in the hematopoietic stem cells, progenitor cells and, except in the megakaryocytic lineage, in the more immature morphologically recognizable precursor cells. The nearly mature blood cells seem to enter the circulation mainly by passing through the endothelial cells of the marrow sinusoids.

Regulation of Hematopoiesis

Hematopoietic stem cells and early progenitor cells show low-level expression of transcription factors and genes specific to several hematopoietic lineages (multilineage priming). Commitment to a single lineage involves enhancement of transcription factors controlling the gene expression programs specific to that lineage and permanent silencing by those transcription factors of gene programs required for differentiation down other lineages.

The mechanisms underlying the commitment of a stem cell to differentiate are not yet fully understood (29,30). According to one model, the probability of a stem cell undergoing self-renewal or differentiation is a stochastic process. Environmental signals (soluble factors, cell-cell and cell extracellular matrix interactions) mediated by specific receptor-ligand interactions operate only by influencing stem cell and progenitor cell apoptosis (and, thus, survival) and proliferation. Another model proposes that all decisions taken by stem cells and progenitor cells are determined by environmental signals. Bone marrow stromal cells (e.g., macrophages, nonphagocytic reticular or fibroblastoid cells, adipocytes, endothelial cells) play a major role in generating such signals; they provide niches for the attachment of stem cells and their progeny, are a source of the extracellular matrix involved in such attachment, and secrete various membrane-bound and soluble stimulatory hematopoietic growth factors and inhibitory cytokines (29,31,32).

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Stem cells and early hematopoietic progenitor cells interact via specific cell surface receptors with multilineage hematopoietic growth factors. The latter include stem cell factor (Steel factor, kit ligand), interleukin-1 (IL-1) and IL-6 for the pluripotent stem cells, and stem cell factor, thrombopoietin, IL-3 (multi-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) for the multipotent myeloid stem cells. The regulation of later progenitor cells and the morphologically recognizable hematopoietic cells is dependent both on multilineage growth factors and lineage-specific growth factors such as G-CSF, M-CSF, IL-5 (influencing CFU-eo), thrombopoietin and IL11 (influencing CFU-mega), and erythropoietin (mainly influencing late BFU-E and CFU-E). The growth factors influencing lymphocyte progenitor cells and precursors include IL-2, IL-4, IL-5, IL-6, IL-7, and IL-11 for the B lineage and IL-2, IL-3, IL-4, IL-7, and IL-10 for the T lineage.

Hematopoietic growth factors are glycoproteins and influence the survival, proliferation, and differentiation of their target cells via second messengers. In their absence, the target cells undergo programmed cell death (apoptosis). Some growth factors such as G-CSF and GM-CSF not only regulate hematopoiesis but also enhance the function of the mature cells. Most hematopoietic growth factors are produced by bone marrow stromal cells and T lymphocytes, either constitutively (e.g., M-CSF production by fibroblastoid cells and endothelial cells) or after their activation by various signals. Thus, fibroblastoid cells and endothelial cells that have been activated by macrophage-derived IL-1 or tumor necrosis factor (TNF) and endotoxin-stimulated macrophages produce M-CSF, GM-CSF, G-CSF, IL-6, and stem cell factor. Antigen- or IL-1 activated T cells produce IL-3, IL-5, and GM-CSF.

The main organ of erythropoietin production in postnatal life is the kidney, and the probable site of synthesis appears to be peritubular cells. About 10% of the erythropoietin is produced in the liver, which is the main organ of synthesis in the fetus. There is an oxygen sensor in the peritubular cells of the kidney, and the production of erythropoietin is inversely proportional to the degree of oxygenation of renal tissue. A limited amount of data suggest that there also may be paracrine or autocrine erythropoietin production in the bone marrow. The erythropoietin receptor is upregulated at the late BFU-E and CFU-E stages, and signalling through this receptor is required to prevent apoptosis.

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In addition to the stimulatory cytokines mentioned above, inhibitors (negative regulators) of hematopoiesis are produced by macrophages, fibroblastoid cells, and endothelial cells. These include transforming growth factor- 1 (TGF- 1), which inhibits multilineage progenitor cells, early erythroid progenitors, and megakaryocytes; TNF- , which inhibits the proliferation of granulocyte precursors; interferon- , which inhibits megakaryocyte progenitors; and macrophage inflammatory protein-1 (MIP-1 ), which inhibits the proliferation of stem cells.

Hematopoiesis in the Embryo and Fetus: Development of the Bone Marrow

Studies in experimental animals have shown that hematopoietic stem cells responsible for embryonic (primitive) hematopoiesis develop in the yolk sac. Those responsible for fetal and postnatal (definitive) hematopoiesis are considered to arise in the aorto-gonad-mesonephros region by some investigators and the yolk sac by others (33,34,35). The stem cells migrate through the blood stream to colonize the fetal liver and other fetal tissues.

In the human embryo, erythropoietic cells first appear within the blood islands of the yolk sac about 19 days after fertilization (36,37). A few megakaryocytes are found in these blood islands during the sixth and seventh weeks of gestation. Yolk sac erythropoiesis is megaloblastic and results in the production of nucleated red cells (Figure 33.2) that contain three embryonic hemoglobins (25), namely, hemoglobins Gower I ( _{2 2}), Gower II ( _{2 2}), and Portland I ( _{2 2}), and, in later embryos, hemoglobin F ( _{2 2}).

Hematopoietic foci develop in the hepatic cords during the sixth week of gestation, and the liver becomes the major site of erythropoiesis in the middle trimester of pregnancy (38,39). During this period, about half the nucleated cells of the liver consist of erythropoietic cells (Figure 33.3). A few granulocytopoietic cells and megakaryocytes also are found in this organ. Fetal hepatic erythropoiesis is normoblastic and gives rise to nonnucleated red cells containing hemoglobin F. These red cells are considerably larger than the red cells of adults. The number of erythropoietic cells in the liver decreases progressively after the seventh month of gestation; a few cells persist until the end of the first postnatal week.

Marrow cavities are formed as a result of the erosion of bone or calcified cartilage by blood vessels and cells from the periosteum (37). The first marrow cavity to develop is that of the clavicle (at about 2 months gestational age). After the formation of the marrow cavities, the vascular connective tissue present within them becomes colonized by circulating hematopoietic stem cells. The latter generate erythropoietic cells during the third and fourth months of gestation, the order of appearance of erythropoietic cells being the same as the order of formation of the marrow cavities. After the sixth month, the bone marrow becomes the major site of hematopoiesis (40). Erythropoiesis in fetal bone marrow is normoblastic and results in the production of nonnucleated red cells, which contain hemoglobins F and A ( _{2 2}) and which are larger than adult red cells. The fetal bone marrow is the predominant site of intrauterine granulocytopoiesis and megakaryocytopoiesis. In this tissue, the myeloid/erythroid ratio (i.e., the ratio of the number of neutrophil precursors plus neutrophil granulocytes to the number of

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Postnatal Changes in the Distribution of Red Marrow and in the Type of Hemoglobin

At birth, all the marrow cavities contain red, hematopoietic marrow. Furthermore, the red marrow contains only a few fat cells. After the first four years of life, an increasing number of fat cells appears between the hematopoietic cells, particularly in certain regions of the marrow, and these regions eventually become yellow and virtually devoid of hematopoietic cells (41,42). Zones of yellow, fatty marrow are found just below the middle of the shafts of the long bones between the ages of 10 and 14 years and, subsequently, extend in both directions, distal spread being more rapid than proximal spread. By the age of about 25 years, hematopoietic marrow is confined to the proximal quarters of the shafts of the femora and humeri, the skull bones, ribs, sternum, scapulae, clavicles, vertebrae, pelvis, and the upper half of the sacrum. Although the distribution of hematopoietic marrow remains essentially unaltered throughout adult life, its fat cell content increases slightly with increasing age and more substantially after the age of 70 years, in association with a gradual expansion of the volume of the marrow cavities.

The percentages of hemoglobins F and A in the blood of full-term neonates are, respectively, 50 to 85% and 15 to 50%. The proportion of hemoglobin F decreases postnatally at different rates in different individuals, but adult levels of less than 1% are reached in nearly all children by the age of 2.5 years.

Because young children have red marrow containing few fat cells in virtually all their marrow cavities, a rapid increase in hematopoietic tissue in this age group is presumably accommodated mainly by a reduction in the proportion of marrow space occupied by sinusoids. If the increase in the rate of hematopoiesis is substantial and prolonged (e.g., in congenital hemolytic anemias), there is an increase in the total volume of the marrow cavities and the reestablishment of extramedullary hematopoiesis in organs such as the liver, spleen, and lymph nodes (43). The expansion of the marrow cavities leads to skeletal abnormalities, such as frontal and parietal bossing, dental deformities, and malocclusion of the teeth. It also causes thinning of the cortex, which may lead to fractures after minor trauma. In adults, increased hematopoiesis is initially associated with the replacement of fat cells in red marrow by hematopoietic cells and also with the spread of red marrow into marrow cavities normally containing yellow marrow (43). If the increase in hematopoiesis is marked, extramedullary hematopoiesis may develop.

Structural Organization of Hematopoietic Marrow

The marrow cavities of most bones contain trabeculae of cancellous bone. The inner surface of the cortex and the outer surfaces of the trabeculae are lined by the endosteum, which consists of a single layer of cells supported on a delicate layer of reticular connective tissue. In most areas of the endosteum, the cells consist of very flat bone-lining cells (endosteal lining cells), but in some areas they consist of osteoblasts or osteoclasts. The marrow, which is located between the trabeculae, is supplied with an extensive microvasculature and some myelinated and nonmyelinated nerve fibers. It does not have a lymphatic drainage (44). The space between the small blood vessels contains a few reticulin fibers and a variety of cell types. The latter include fat cells, precursors of red cells, granulocytes, monocytes and platelets, lymphocytes, plasma cells, macrophages (phagocytic reticular cells), nonphagocytic reticular cells, and mast cells (4,45).

Blood Supply

One or more nutrient canals penetrate the shafts of the long bones obliquely. Each canal contains a nutrient artery and one or two nutrient veins. After entering the marrow, the nutrient artery divides into ascending and descending branches, which coil around the central longitudinal vein, the main venous channel of the marrow. The ascending and descending arteries give off numerous arterioles and capillaries that travel radially toward the endosteum and often open into a plexus of sinusoids (25). The sinusoids drain through a system of collecting venules and larger venous channels into the central longitudinal vein, which in turn drains mainly into the nutrient veins. In the diaphyses of long bones containing yellow fatty marrow, the nutrient artery gives off relatively few branches until it reaches the lower edge of the red marrow, where it breaks up into numerous vessels that penetrate the hematopoietic tissue. Many blood vessels of various sizes supply the marrow within flat and cuboidal bones, entering the marrow cavity via one or more large nutrient canals, as well as through numerous smaller canals.

There are interconnections between the blood supply of the bone marrow and bone through an endosteal network of blood vessels. This network communicates both with the periosteal vessels via fine veins passing through the bone and with branches of the nutrient artery. Furthermore, studies in experimental animals have shown that many capillaries derived from the nutrient artery enter Haversian canals but swing back into the marrow and open into sinusoids or venules (46,47). There has been much speculation as to whether blood reaching the marrow from the bone contains one or more

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The sinusoids of human bone marrow have thin walls consisting of an inner complete layer of flattened endothelial cells with little or no underlying basement membrane and an outer incomplete layer of adventitial cells (48). The endothelial cells are characterized by the presence of numerous small pinocytotic vesicles along both their luminal and abluminal surfaces (Figure 33.4). The nucleus is flattened and contains moderate quantities of nuclear membrane associated condensed chromatin. The cytoplasm also contains ribosomes, rough endoplasmic reticulum (RER), mitochondria, some microfilaments, a few lysosomes, and occasional fat droplets. Adjacent endothelial cells overlap and may interdigitate extensively. These areas of contact are characterized by (a) a strictly parallel alignment of the membranes of the interacting cells with a narrow gap between the opposing membranes and (b) short stretches in which the membranes fuse together, forming tight junctions (not true desmosomes). There is an increased electron density of the cytoplasm immediately adjacent to and on both sides of the tight junctions (Figure 33.4). Some endothelial cells show alkaline phosphatase activity. Endothelial cells contain no stainable iron except when the iron stores are increased. They produce extracellular matrix, stem cell factor, IL6, GM-CSF, IL-1 , IL-11, and G-CSF and are thus intimately involved with the regulation of hematopoiesis. Endothelial cells of the sinusoids allow the bidirectional migration of progenitor cells and hematopoietic stem cells through them by a mechanism involving specific binding molecules.

Adventitial cells project long peripheral cytoplasmic processes, which may be closely associated with some extracellular reticulin fibers. Some of these processes lie along the sinusoidal surface, and others protrude outward between hematopoietic cells. Thus, adventitial cells are a type of reticular cell (i.e., form part of the cytoplasmic network or reticulum of the marrow stroma). The cytoplasm of adventitial cells contains ribosomes, RER, some pinocytotic vesicles, a few electron-dense lysosomes, occasional fat globules, and numerous microfilaments that are often arranged in bands. The latter are usually situated within the peripheral cytoplasmic processes. The cytoplasm of some adventitial cells appears very electron lucent. Adventitial cells stain strongly for alkaline phosphatase.

Nerve Supply

In the case of a long bone, the nerve supply enters the bone marrow mainly via the nutrient canal but also through a number of epiphyseal and metaphyseal foramina. Bundles of nerve fibers travel together with the nutrient artery and its branches and supply the smooth muscle in such vessels or, occasionally, terminate between hematopoietic cells (49).

Extracellular Matrix (Connective Tissue)

Normal marrow contains a scanty incomplete network of fine branching reticulin fibers between the parenchymal cells (Figure 33.5). A higher concentration of thicker fibers is found in and around the walls of the larger arteries and

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Stromal Cells

The stromal cells comprise (a) osteoblasts, bone marrow fat cells (adipocytes), and nonphagocytic reticular cells (including myofibroblasts), all of which are derived from mesenchymal stem cells within the marrow; (b) osteoclasts, macrophages, and mast cells that are derived from the myeloid hematopoietic stem cell; and (c) endothelial cells (discussed above) that are derived either from the hematopoietic stem cell or a more primitive marrow cell that also gives rise to hematopoietic stem cells (28). Some stromal cells are intimately involved in the regulation of hematopoiesis.

Osteoblasts and Osteoclasts

Osteoblasts are present in the endosteum in areas of deposition of osseous matrix (osteoid). In histologic sections, osteoblasts are cuboidal or pyramidal and have eccentric nuclei. Their cytoplasm is markedly basophilic and contains a large round pale zone. Osteoblasts are frequently found in a continuous layer, usually one or two cells thick, and appear like an area of epithelium. They become surrounded by the osteoid they produce and thus eventually become osteocytes. Osteoclasts are large multinucleate cells involved in bone resorption and are often found in shallow excavations on the surface of the bone, termed Howship's lacunae. Osteoblasts arise from progenitor cells closely associated with the endosteal lining cells. Although it is usually considered that osteoblast progenitor cells are not derived from hematopoietic stem cells, recent studies in mice indicate that osteoblasts and hematopoietic cells arise from a common primitive marrow cell (50). Osteoblasts produce cytokines such as IL6, G-CSF, and GM-CSF that influence hemopoiesis (51). Osteoclasts originate from the myeloid hematopoietic stem cells. The relationship between the osteoclast progenitor cell and other hematopoietic progenitor cells (e.g., CFU-GEMM, CFU-GM, CFU-M) is not yet clear (52).

Romanowsky-stained normal marrow smears may contain groups of osteoblasts or individual osteoclasts. In such preparations, osteoblasts have an oval or elongated shape and are 20 to 50 m in diameter. They have abundant basophilic cytoplasm, often with somewhat indistinct margins, and a single small eccentric nucleus with only small quantities of condensed chromatin and with one to three nucleoli. The cytoplasm contains a rounded pale area corresponding to the Golgi apparatus, which often is situated some distance from the nucleus (Figure 33.6). Osteoblasts stain positively for alkaline phosphatase activity. They superficially resemble plasma cells, but the latter are smaller, contain heavily stained clumped chromatin, and have a Golgi zone situated immediately adjacent to the nucleus. Osteoclasts appear as giant multinucleate cells with abundant pale blue cytoplasm containing many azurophilic (purple-red) granules (Figure 33.7). The individual nuclei are rounded in outline, uniform in size, contain a single prominent nucleolus, and do not overlap. Osteoclasts are strongly acid phosphatase positive. They must be distinguished from the other polyploid giant cells in the marrow, the megakaryocytes. These are usually not multinucleate but contain a single large lobulated nucleus.

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Fat Cells

The number of fat cells in hematopoietic bone marrow varies markedly with age (53,54). In normal adults, 30 to 70% of the area of a histologic section of hematopoietic marrow consists of fat cells (Figures 33.8,33.9). Fat cells are the largest cells in the marrow, and sections of such cells have average diameters of about 85 m. Ultrastructural studies show that these cells have a single large fat globule at their center and a narrow rim of cytoplasm at their periphery. This cytoplasmic rim contains a flattened nucleus, several small lipid droplets, ribosomes, strands of endoplasmic reticulum, and several mitochondria. The fat cells of the bone marrow only have small quantities of reticulin and collagen fibers around them. They are in intimate contact with vascular channels, macrophages, and all types of hematopoietic cells. Marrow fat cells seem to be formed by the accumulation of lipid within adventitial cells, other nonphagocytic reticular cells, and, possibly, sinus endothelial cells. Whenever there is an increase or decrease in the number of hematopoietic cells in bone marrow, there is a corresponding decrease or increase, respectively, of the number of fat cells so that the intersinusoidal space within marrow cavities is always fully occupied by cells. The mechanisms underlying this inverse relationship between the mass of fat cells and hematopoietic cells in the marrow are uncertain. In severe anorexia nervosa or cachexia secondary to chronic disorders, such as tuberculosis or carcinoma, there is a marked reduction in fat cells, often together with a reduction in hematopoietic tissue. In these conditions, the space normally occupied by cells is filled with a gelatinous extracellular substance composed of acid mucopolysaccharide (55).

Macrophages (Phagocytic Reticular Cells)

The bone marrow contains many macrophages. The frequency of this cell type is best appreciated in sections of trephine biopsies stained for an antigen found in macrophages such as CD68 (Figure 33.10) or in electron micrographs of ultrathin sections of marrow fragments rather than in smears of aspirated bone marrow. In H&E-stained sections of trephine biopsies, macrophages appear as moderately large cells with abundant cytoplasm. In Romanowsky-stained marrow smears, they appear as irregularly shaped cells 20 to 30 m in diameter and have a round or oval nucleus with pale, lacelike chromatin and one or more large nucleoli. The cytoplasm is voluminous, stains pale blue, and contains azurophilic granules, vacuoles, and variously sized inclusions consisting of

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In unstained smears and sections of normal marrow and in Giemsa- or H&E-stained sections, macrophages may show refractile yellow-brown hemosiderin-containing intracytoplasmic inclusions, which vary between 0.5 and 4 m in diameter. These appear as blue or blue-black granules when stained by Perls' acid ferrocyanide method. This stain also may color the entire cytoplasm a diffuse pale blue (Figure 33.12). The amount of iron-positive granules within the marrow fragments on a marrow smear (Figure 33.13) or the amount in a histologic section of a trephine biopsy sample may be assessed semiquantitatively and is a useful guide to the total iron stores in the body (56). Stainable iron is absent or virtually absent in iron deficiency (with or without anemia) and increased in conditions such as hereditary hemochromatosis or transfusion-induced iron overload. Macrophages contain PAS-positive material and are strongly positive for -naphthyl acetate esterase (Figure 33.11B C)

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Ultrastructural studies of marrow fragments show that macrophages form long cytoplasmic processes at their periphery and that such processes extend for considerable distances between various types of hematopoietic cells (Figure 33.14). Some cytoplasmic processes protrude through the endothelial cell layer into the sinusoidal lumen (Figure 33.15) and appear to be involved in recognizing and phagocytosing circulating microorganisms and senescent or damaged erythrocytes and granulocytes. The nucleus often has an irregular outline and contains small to moderate quantities of nuclear membrane associated condensed chromatin. The cytoplasm has many strands of RER, scattered ferritin molecules, a well-developed Golgi apparatus, several mitochondria, a number of small or medium-sized homogeneous electron-dense primary lysosomes of variable shape, and a number of large inclusions. Some of the latter have a complex ultrastructure with both electron-dense and electron-lucent areas and myelin figures and may contain numerous ferritin and hemosiderin molecules; these appear to represent secondary lysosomes with residual material from phagocytosed cells (Figure 33.16). Other large inclusions can be recognized readily as granulocytes (Figure 33.17), extruded erythroblast nuclei, and erythrocytes at various stages of degradation. A few reticulin fibers may be found in contact with parts of the cell surface.

Macrophages are present within erythroblastic islands (Figure 33.11C), plasma cell islands, and lymphoid nodules but also may occur elsewhere in the marrow parenchyma. Some are found immediately adjacent to the endothelial cells of sinusoids, forming part of the adventitial cell layer. Bone marrow macrophages not only function as phagocytic cells but also generate various hematopoietic growth factors

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Nonphagocytic Reticular Cells

In Romanowsky-stained marrow smears, nonphagocytic reticular cells have an irregular or spindle shape and resemble macrophages except that they lack large intracytoplasmic inclusions. Light microscope cytochemical and histochemical data indicate that these cells are PAS-negative, strongly positive for alkaline phosphatase, negative for acid phosphatase, negative or only weakly positive for -naphthyl acetate esterase, and negative for stainable iron. Thus, there seems to be some overlap between the cytochemical characteristics of nonphagocytic reticular cells and macrophages (57,58). In the case of mice and rats, however, light and electron microscopic cytochemical data have clearly established the existence of two distinct types of reticular cells in the marrow stroma: (a) fibroblast-like nonphagocytic reticular cells that have cell membrane associated alkaline phosphatase and no acid phosphatase and (b) macrophage-like phagocytic reticular cells that are positive for acid phosphatase but not for alkaline phosphatase (59).

Electron microscopic studies of nonphagocytic reticular cells in human bone marrow (48,60,61) have shown that, like macrophages, these cells extend branching cytoplasmic processes between hematopoietic cells and are in contact with extracellular reticulin fibers (Figure 33.18). However, unlike macrophages, they do not have secondary lysosomes or have only an occasional secondary lysosome. They may contain variable numbers of filaments or a few small fat globules in their cytoplasm. The intracytoplasmic filaments sometime occur in bundles, and the cells are then ultrastructurally indistinguishable from adventitial cells. It is possible that the nonphagocytic reticular cells comprise a number of different cell types including fibroblasts or myofibroblasts, adventitial cells, and cells whose functions have not yet been defined. Myeloid cell and B-lymphoid progenitors are located adjacent to myofibroblasts.

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At least some of the nonphagocytic reticular cells arise from a mesenchymal stem cell capable of giving rise to colonies of fibroblast-like or myofibroblast-like cells in vitro. As mentioned earlier, nonphagocytic reticular cells appear to play an important role in the microenvironmental regulation of hematopoiesis, both by binding to primitive hematopoietic cells (62) and by producing certain hematopoietic growth factors both constitutively and in response to stimulation by monokines (63). In mice and presumably also in humans, they synthesize collagen (types I and III) and fibronectin.

Mast Cells

Mast cells tend to be found in association with the periphery of lymphoid follicles and the adventitia of small arteries and adjacent to the endosteal cells of bone trabeculae and the endothelial cells of sinusoids.

It is now known that the hematopoietic stem cells generate morphologically unrecognizable progenitors of mast cells within the bone marrow (64) and that the most mature of these cells enter the blood (65,66). The circulating cells, which still lack mast cell granules, migrate into the tissues, where they proliferate and mature into mast cells. Mast cells and basophils appear not to share a common early progenitor cell (67).

Unlike the granules of basophils, which are very water soluble, those of mast cells are much less so. Nevertheless, mast cells are not easily recognized in sections of marrow stained with H&E. By contrast, they are readily identified in sections stained with the Giemsa stain. In such sections, mast cells have round, oval, or spindle-shaped outlines and many dark purple cytoplasmic granules. The nucleus is often oval and may be situated eccentrically. Immunochemical staining can be performed using the antibody AA1, which reacts with mast cell tryptase (10) and does not cross-react with basophils. In Romanowsky-stained marrow smears, mast cells vary between 5 and 25 m in their long axis and tend to have an ovoid or elongated shape (Figure 33.19A B). The cytoplasm is packed with coarse purple-black to red-purple granules; but, unlike in basophil granulocytes, the granules seldom overlie the nucleus. The nucleus is small, round or oval, and either centrally or eccentrically located. It contains less condensed chromatin than that of a basophil granulocyte. The granules of mast cells are rich in heparin and stain metachromatically with toluidine blue. Mast cells are also peroxidase negative, PAS positive, acid phosphatase positive, and -naphthol AS-D chloroacetate esterase positive. Unlike basophil granulocytes, mast cells are capable of mitosis.

In the electron microscope, the granules of mast cells vary considerably in appearance. They may be homogeneously electron dense, have areas of increased electron density at their centers, or contain parallel arrangements, whorls, or scrolls of a crystalline or fibrillar structure (Figures 33.20A B). The nucleus contains moderate quantities of condensed chromatin. In addition to the numerous granules, the cytoplasm contains some mitochondria, a few short strands of endoplasmic reticulum, occasional lipid droplets, and some fibrils.

Hematopoietic Cells

Lymphocytes and Plasma Cells

Histologic sections of normal marrow show nodules of small lymphocytes that are 0.08 to 1.2 mm in diameter and contain occasional reticular cells (68) (Figure 33.21A), as well as much smaller lymphoid aggregates; these nodules and aggregates are usually found in the intertrabecular and perivascular areas and are rarely found immediately adjacent to trabeculae. Paratrabecular nodules and aggregates are characteristic of infiltration by malignant lymphoid cells. In normal marrow, the prevalence of lymphoid nodules increases with age. About 20% of such nodules are irregular in outline, poorly circumscribed, and often contain several fat cells and a few eosinophils between the lymphocytes; they do not contain germinal centers, and their

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T and B lymphocytes (with more T than B cells) and plasma cells also are found unassociated with lymphoid nodules. Lymphocytes are found scattered between hematopoietic cells, and plasma cells are often present in small groups, surrounding a central macrophage or sheathing some small blood vessels.There are more than light chain positive plasma cells (Figure 33.22A B).

Precursors of Red Cells, Granulocytes, Monocytes, and Platelets

The early granulocytopoietic cells (myeloblasts and promyelocytes) mainly are found near the endosteum of bone trabeculae and the adventitial aspects of arterioles (Figure 33.22C). Maturing granulocyte precursors radiate outward from these sites, and the neutrophil granulocytes often are found in the center of intertrabecular areas, adjacent to sinusoids. A few promyelocytes and myeloctyes are present singly or in small clusters at sites away from bone trabeculae and blood vessels. Erythroblasts and megakaryocytes are also found away from the endosteum. The erythroblasts occur in one or two layers surrounding one or two central macrophages; the late erythroblasts and marrow reticulocytes usually are situated next to sinusoids. The megakaryocytes usually lie near sinusoids (Figure 33.22D) and protrude cytoplasmic processes into their lumina; these processes discharge platelets directly into the microcirculation.

Hematopoietic Cells: Characteristics in Marrow Smears and Ultrastructure

Neutrophil Precursors

The earliest morphologically recognizable neutrophil precursor is termed the myeloblast. The successive cytologic

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A myeloblast is 10 to 20 m in diameter. It has a large rounded nucleus with finely dispersed chromatin and two to five nucleoli. The nucleus-to-cytoplasm ratio is moderately high, and the cytoplasm is basophilic and nongranular. It is likely that only some myeloblasts mature into neutrophil promyelocytes and that others mature into eosinophil or basophil promyelocytes.

Neutrophil promyelocytes are larger than myeloblasts and have basophilic cytoplasm containing a few to several purple-red (azurophilic) granules. The nuclear chromatin pattern is slightly coarser than in myeloblasts, and there may be prominent nucleoli. The neutrophil myelocytes are characterized by the presence in their cytoplasm of many fine light pink (neutrophilic) granules in addition to some azurophilic granules; the neutrophilic granules also are termed specific granules. The nucleus is often eccentric and is round, oval, or slightly indented. The nuclear chromatin is coarsely granular, and the nucleoli are indistinct. The cytoplasm occupies a larger fraction of the cell volume than in promyelocytes; it initially appears pale blue but subsequently becomes predominantly pink. The progressive reduction of cytoplasmic basophilia during the maturation of a myeloblast to a mature myelocyte results largely from a reduction of blue-staining cytoplasmic RNA. The neutrophil metamyelocyte has a C-shaped nucleus and an acidophilic cytoplasm containing numerous fine neutrophilic granules. Few or no azurophilic granules are seen. Juvenile neutrophils (also called band or stab forms) have U-shaped or long, relatively narrow, bandlike nuclei that are often twisted into various configurations. The nuclei contain large clumps of condensed chromatin and may show one or more partial constrictions along their length. These constrictions become progressively more complete and eventually develop into the fine strands of chromatin that are typical of the segmented nuclei of marrow and blood neutrophil granulocytes. Most neutrophil granulocytes have two to five nuclear segments that are joined together by such strands. Some of the neutrophil granulocytes of females have a drumsticklike nuclear appendage (representing an inactivated X chromosome) attached to one of the nuclear segments.

Cytochemistry

When stained by the PAS reaction, myeloblast cytoplasm shows a diffuse, pale red-purple tinge, sometimes with fine granules of the same color. Myeloblasts either do not stain with Sudan black or show a few small sudanophilic granules near the nucleus. They are also peroxidase-negative and, usually, -naphthol AS-D chloroacetate esterase negative. The cytoplasm of neutrophil promyelocytes and

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Ultrastructure

Myeloblasts show no special ultrastructural features (72,73,74,75). The nucleus has one or more well-developed nucleoli and shows only slight peripheral chromatin condensation. The cytoplasm contains many ribosomes but only a few strands of endoplasmic reticulum and a poorly developed Golgi apparatus. By contrast, the cytoplasm of a promyelocyte is much more complex, being rich in ribosomes, RER, and mitochondria. It also contains a highly developed Golgi apparatus. During the maturation of a promyelocyte to a neutrophil granulocyte, there is a progressive increase in the degree of condensation of nuclear chromatin; a progressive reduction in the quantity of ribosomes, RER, and mitochondria; a diminution of the Golgi apparatus after the myelocyte stage; and the accumulation of large quantities of glycogen at the metamyelocyte and

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The primary granules observed with the electron microscope correspond to the azurophilic granules seen in Romanowsky-stained smears, and the secondary and tertiary

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Eosinophil and Basophil Precursors

The eosinophil and basophil granulocytes develop through stages that are essentially similar to those through which the neutrophil granulocytes develop. The earliest morphologically recognizable precursors are cells in which a few eosinophil or basophil granules have formed, that is, the eosinophil promyelocytes and basophil promyelocytes. Eosinophil promyelocytes have rounded nuclei with dispersed chromatin and nucleoli and contain two types of granules: large red-orange (eosinophilic) granules and large bluish granules. Eosinophil myelocytes (Figure 33.29), metamyelocytes, and granulocytes have only large eosinophilic granules. Basophil myelocytes, metamyelocytes, and granulocytes are characterized by the presence of large, round, deeply basophilic granules that often overlie the nucleus (Figure 33.30); the more mature granules stain metachromatically with toluidine blue. The majority of circulating eosinophil and basophil granulocytes have two nuclear segments.

Cytochemistry

The granules of eosinophil and basophil granulocytes and their precursors do not stain by the PAS reaction (24,76). However, PAS-positive deposits are found between the specific granules in both cell lineages. The periphery of the eosinophil granules of all cells of the eosinophil series stains strongly with Sudan black, and the core stains weakly or not at all. Basophil granules are strongly sudanophilic in basophil promyelocytes and myelocytes, but the degree of sudanophilia decreases with increasing maturity; in mature basophils, the granules either do not stain or stain metachromatically (reddish). Peroxidase and acid phosphatase but not lysozymes are demonstrable in the eosinophil granules in all eosinophil precursors and eosinophils. Human eosinophil peroxidase is biochemically and immunochemically distinct from myeloperoxidase, the type of peroxidase present in the neutrophil series. In the basophil series, the granules are strongly positive for peroxidase in basophil promyelocytes and myelocytes, weakly positive in basophil metamyelocytes, and almost negative in basophil granulocytes. Basophil granules stain positively for acid phosphatase. Basophil and eosinophil granulocytes are essentially negative for -naphthol AS-D chloroacetate esterase and -naphthyl butyrate esterase.

Eosinophil granules contain eosinophil cationic proteins and an arginine- and zinc-rich major basic protein that are involved in the killing of metazoan parasites. The major basic protein also stimulates basophils and mast cells to release histamine. Other constituents of eosinophil granules include histaminase and arylsulfatase, which are involved in the modulation of immediate-type hypersensitivity reactions. Basophil granules contain chondroitin sulfate and heparin sulfate, which account for their property of staining metachromatically (red-violet) with toluidine blue. They also contain histamine, one of the substances released when immunoglobulin E (IgE)-coated basophils react with specific antigen.

Ultrastructure

On the basis of their electron microscopic features, two types of eosinophil granules, termed primary and secondary granules, are recognized (75,77). Primary granules are large, rounded, homogeneous, and electron dense, and

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Cells of the basophil series contain characteristic basophil granules, which are prone to undergo varying degrees of extraction during processing for electron microscopy (Figure 33.33). Basophil granules are made up of numerous, closely packed, fine rounded particles (Figure 33.34); the particles are about 20 nm in diameter in mature basophils and slightly smaller in basophil promyelocytes and myelocytes.

Monocyte Precursors

The morphologically recognizable cells belonging to the monocyte series are the monoblasts, promonocytes, marrow monocytes, and blood monocytes. The blood monocytes are not end cells but develop further in the tissues to become macrophages. Certain data suggest that macrophages and osteoclasts have a common progenitor. All these cells are considered to constitute the mononuclear phagocyte system. In this system, cell division occurs mainly in the monoblasts and promonocytes.

Monoblasts are similar in appearance to myeloblasts except that their nuclei may be slightly indented or lobulated.

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Marrow monocytes and blood monocytes have a lower nucleus-to-cytoplasm ratio (<1), a less basophilic cytoplasm, and a larger number of azurophilic granules than promonocytes. The cytoplasm is pale gray-blue, has a ground glass appearance, and may contain vacuoles. The nucleus is eccentrically placed and may be oval, kidney-shaped, horseshoe-shaped, or lobulated; the chromatin has a skeinlike or lacy appearance.

Cytochemistry

Some normal monocytes show several fine or moderately coarse PAS-positive granules and sudanophilic granules and a few peroxidase-positive granules scattered in their cytoplasm (24,71,78). Monocytes do not stain for alkaline phosphatase but stain strongly for acid phosphatase. They contain lysozyme.

Monocytes are -naphthol AS-D chloroacetate esterase negative but are -naphthyl acetate esterase (nonspecific esterase) positive. Alpha-naphthyl acetate esterase activity is present not only in monocytes and macrophages, but also in other myeloid cells, including neutrophil promyelocytes and myelocytes, megakaryocytes, and immature red cell precursors. Alpha-naphthyl butyrate esterase activity is stronger than -naphthyl acetate esterase activity in monocytes and macrophages and is much weaker in the other types of myeloid cells mentioned above. Both the -naphthyl acetate and the -naphthyl butyrate esterase activities of monocytes are inhibited by fluoride; in granulocytes and their precursors, these enzyme activities are fluoride insensitive.

Ultrastructure

The earliest monocyte precursor that can be identified on ultrastructural criteria (72,75) is the promonocyte. The nucleus of this cell has only small quantities of nuclear membrane associated condensed chromatin and has one or more nucleoli. The cytoplasm contains many ribosomes, a moderate number of mitochondria, several strands of RER, bundles of fibrils, a prominent Golgi apparatus, and a few characteristic cytoplasmic granules. The strands of endoplasmic reticulum are shorter and less abundant than in neutrophil promyelocytes. Two types of cytoplasmic granules are seen in promonocytes: (a) immature granules, which have a central zone of flocculent electron-dense material and a clear peripheral zone, and (b) mature granules, which are smaller than the immature granules, vary considerably in size and shape, and are homogeneously electron dense (Figures 33.36,33.37). The maturation of promonocytes first into marrow monocytes and then into blood monocytes is associated with some increase in the quantity of condensed chromatin in the nucleus, a progressive reduction in the number of ribosomes, RER, and fibrils in the cytoplasm, and an increase in the number of cytoplasmic granules. Most or all of the granules of marrow monocytes and all the granules of blood monocytes are of the mature type. Ultrastructural cytochemical studies have shown that some large round granules have acid phosphatase activity and that such granules are more frequent in promonocytes than

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Red Cell Precursors

In this chapter, the term erythroblast is used to describe any nucleated red cell precursor, normal or pathologic, and the term normoblast to describe all cells that have the morphologic characteristics of the erythroblasts found in normal bone marrow. The terms used to describe various classes of normal red cell precursor are, in order of increasing maturity, pronormoblast, basophilic normoblast, early polychromatic normoblast, late polychromatic normoblast, marrow reticulocyte, and blood reticulocyte (Figure 33.38). Cell division occurs only in the first three of these cytologic classes. Marrow samples containing normoblasts are said to show normoblastic erythropoiesis.

Pronormoblasts are large cells with a diameter of 12 to 20 m. They have rounded nuclei and moderate quantities of agranular cytoplasm that stains intensely basophilic except for a pale area (corresponding to the Golgi apparatus) adjacent to the nucleus. The nuclear chromatin has a finely stippled or fine reticular appearance, and there are one or more prominent nucleoli. The basophilic normoblasts resemble pronormoblasts except that their nuclear chromatin is slightly more condensed and consequently has a coarsely granular appearance. The early polychromatic normoblasts are smaller than basophilic normoblasts and have a smaller nucleus and a lower nucleus-to-cytoplasm ratio. The cytoplasm is polychromatic and agranular, and the nucleus contains several medium-sized clumps of condensed chromatin, particularly adjacent to the nuclear membrane. The polychromasia results from the presence of moderate quantities of cytoplasmic RNA (which stains blue), as well as of hemoglobin (which stains red). Late polychromatic normoblasts are even smaller and show a further reduction in the ratio of the area of the nucleus to the area of the cytoplasm. The cytoplasm is predominantly orthochromatic but still has a grayish tinge (i.e., is faintly polychromatic). The nucleus is small and eccentric and contains large clumps of condensed chromatin. The nuclear diameter is less than about 6.5 m. When mature, late polychromatic normoblasts extrude their nuclei and become marrow reticulocytes; the extruded nuclei are rapidly phagocytosed and degraded by adjacent macrophages. The marrow reticulocyte is irregular in outline and has faintly polychromatic cytoplasm. It is motile and soon enters the marrow sinusoids. When marrow and blood reticulocytes are stained supravitally with brilliant cresyl blue, the ribosomal RNA responsible for their polychromasia precipitates into a basophilic reticulum (hence the term reticulocyte). Reticulocytes circulate in the blood for one to two days before becoming mature red cells. The average volume of blood reticulocytes is 20% larger than that of red cells. The latter are circular, biconcave, and acidophilic (i.e., stain red) and, in dried fixed smears, have an average diameter of 7.2 m (range: 6.7 7.7 m).

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Cytochemistry

Normal erythroblasts are PAS negative. They also fail to stain with Sudan black and are peroxidase negative. Most nucleated red cells are -naphthol AS-D chloroacetate esterase negative, but occasional cells show a few positive granules. A few -naphthol butyrate esterase positive granules are seen in some nucleated red cells of all degrees of maturity; the positive granules are sometimes seen at the nuclear margin. Coarse acid phosphatase positive paranuclear granules are frequently present in all types of erythroblasts.

In normal bone marrow smears stained by Perls' acid ferrocyanide method, 20 to 90% of the polychromatic erythroblasts contain one to five small blue-black granules that are usually just visible at high magnification (Figure 33.38C). These iron-containing (siderotic) granules are randomly distributed within the cytoplasm and correspond to the siderosomes seen under the electron microscope. Erythroblasts containing siderotic granules are termed sideroblasts. In iron deficiency anemia and, to a lesser extent, in the anemia of chronic disorders, the percentage of sideroblasts is decreased. In conditions associated with an increased percentage saturation of transferrin (e.g., hemolytic anemias), the average number of siderotic granules per cell and the average size of such granules are increased.

Ultrastructure

All nucleated red cell precursors are characterized by the presence of small surface invaginations that develop into intracytoplasmic vesicles (rhopheocytotic vesicles) (75) (Figure 33.39). The nucleus of the pronormoblast has a small quantity of nuclear membrane associated condensed chromatin (Figure 33.40). The cytoplasm is of low-electron density and contains numerous ribosomes, a moderately well-developed Golgi apparatus, several mitochondria, some strands of endoplasmic reticulum, and small numbers of scattered ferritin molecules. It also contains a few pleomorphic electron-dense acid phosphatase positive lysosomal granules, which are usually arranged in a group near the Golgi saccules. During the maturation of a pronormoblast into a late polychromatic normoblast (Figure 33.41), the following changes are seen: (a) a steady increase in the quantity of condensed chromatin, (b) a gradual increase in the electron density of the cytoplasmic matrix due to the synthesis of increasing quantities of hemoglobin, (c) a progressive reduction in the number of ribosomes in the cytoplasm, (d) a reduction in the number and size of the mitochondria, and (e) an increasing tendency for some of the intracytoplasmic ferritin molecules to aggregate and form siderosomes (Figures 33.42,33.43). Small autophagic

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Dyserythropoiesis and Ineffective Erythropoiesis

Most of the erythroblasts in normal bone marrow are uninucleate and do not display any unusual morphologic features. However, when 400 to 1,000 consecutive erythroblasts (excluding mitoses) were studied in bone marrow smears from each of 10 healthy volunteers with stainable iron in the bone marrow, 0 to 0.57% (mean: 0.31%) were found to be binucleate, 0.7 to 4.8% (mean: 2.4%) showed intererythroblastic cytoplasmic bridges, 0 to 0.9% (mean: 0.24%) showed cytoplasmic stippling, and 0 to 0.7% (mean: 0.39%) showed cytoplasmic vacuolation. In addition, 0 to 0.55% (mean: 0.22%) had markedly irregular nuclear outlines or karyorrhectic nuclei, and 0 to 0.39% (mean: 0.18%) contained Howell-Jolly bodies (micronuclei), a marker of chromosome breaks (80) (Figure 33.46). In another study of 15 healthy males in which 5,000 erythroid cells (including mitoses) were assessed per subject, 0.14% 0.04 (SD) were found to be binucleate or multinucleate cells or to be pluripolar mitoses (81). A number of other unusual morphologic features are seen in some erythroblast profiles when the marrow is examined with the electron microscope. These include short stretches (250 910 nm) of duplication of the nuclear membrane in 2% of the profiles, short (260 520 nm) intranuclear clefts in 1.7%, and iron-laden mitochondria in less than 0.2% (79). The above-mentioned light and electron microscopic features are sometimes described as dyserythropoietic changes, with the implication that they are morphologic manifestations of a minor disturbance of

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The phrase ineffective erythropoiesis is used to describe the loss of potential erythrocytes due to the phagocytosis and destruction of developing erythroblasts within the bone marrow. The extent of ineffective erythropoiesis in normal bone marrow is small (25). In a number of conditions such as homozygous -thalassemia and the megaloblastic anemias, there is a gross increase in the ineffectiveness of erythropoiesis; some of the abnormal erythroblasts undergo apoptosis prior to phagocytosis. In such conditions, erythroblasts at various stages of degradation may be recognized within marrow macrophages, both by light and electron microscopy. Apoptosis at the late BFU-E and CFU-E stages is thought to be a major factor controlling the rate of erythropoiesis.

Megakaryocytes

The majority of the cells of the megakaryocyte series are larger than other hematopoietic cells and have polyploid DNA contents. The earliest morphologically recognizable cells in this series are called megakaryoblasts. These are 20 to 30 m in diameter and have a single large, oval, kidney-shaped, or lobed nucleus that is surrounded by a narrow rim of intensely basophilic agranular cytoplasm. The nucleus contains several nucleoli. Megakaryoblasts (group I megakaryocytes) mature into promegakaryocytes (group II megakaryocytes), which in turn develop into granular megakaryocytes (group III megakaryocytes). Promegakaryocytes are larger than megakaryoblasts and have a larger volume of cytoplasm relative to that of the nucleus. They possess a single large multilobed nucleus with the overlapping lobes arranged in a C-shaped formation. The cytoplasm is less basophilic than that of megakaryoblasts and contains a few azurophilic granules that are usually grouped within the concavity formed by the overlapping nuclear lobes. The granular megakaryocytes (Figure 33.47) are up to 100 m in diameter and have abundant pale-staining cytoplasm containing many azurophilic granules. The nucleus has multiple lobes, and these become

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Mature platelets are usually 2 to 3 m in diameter and are irregular in outline. The cytoplasm stains pale blue and has a number of azurophilic granules at its center. Newly formed platelets are slightly larger than mature ones.

About 40% of megakaryoblasts, 20% of promegakaryocytes, and 2% of granular megakaryocytes synthesize DNA (82). However, cell division is probably uncommon in megakaryoblasts and is not seen in the other two cell types. The occurrence of cycles of DNA replication without cytokinesis results in the characteristic polyploidy of these cells. The total DNA contents of megakaryoblasts range between 4c and 32c and of promegakaryocytes and granular megakaryocytes between 8c and 64c (1c = the haploid DNA content). There is a positive correlation between the nuclear area and DNA content of megakaryocytes.

Cytochemistry

When stained by the PAS reaction, megakaryocytes show a diffuse and finely granular positivity over both the nucleus and the perinuclear and intermediate zones of the cytoplasm (24,69,70,71). These positive areas also contain varying numbers of densely positive blocks (Figure 33.48). A narrow peripheral zone of the cytoplasm is often PAS negative, and this may be surrounded by clumps of positive granules within attached platelets. Within platelets, PAS-positive material appears as scattered, lightly staining fine granules at the periphery and as clumps of darkly staining coarse granules at the center. Megakaryocytes and platelets are usually unstained by Sudan black, but occasional megakaryocytes may show a diffuse positivity with fine positive granules scattered both in the cytoplasm and over the nucleus. Megakaryocytes and platelets display strong acid phosphatase activity.

Peroxidase activity cannot be demonstrated in megakaryocytes by light microscopy but can be demonstrated in a characteristic distribution using the electron microscope.

Megakaryocytes show no -naphthol AS-D chloroacetate esterase activity. However, they have substantial -naphthyl acetate esterase activity (Figure 33.49) and weaker -naphthyl butyrate esterase activity; the latter generates many coarse or fine positive granules in the cytoplasm and over the nucleus.

Ultrastructure

The nucleus of a megakaryoblast has two or more lobes, very little condensed chromatin, and prominent nucleoli (75,83,84). The cytoplasm contains large numbers of ribosomes, scattered RER, several mitochondria, and a few

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Three zones can be recognized in the extensive cytoplasm of a granular megakaryocyte (Figure 33.50): (a) a narrow perinuclear zone containing the Golgi apparatus and some of the ribosomes, RER, and mitochondria, (b) a wide intermediate zone containing many ovoid, electron-dense a granules, numerous sacs of the DMS, lysosomal vesicles, ribosomes, RER, and mitochondria, and (c) a narrow outer zone that is devoid of organelles. Mature granular megakaryocytes protrude cytoplasmic processes that lie near to or within marrow sinusoids. Platelets are formed by the fragmentation of these processes, the platelet membranes being made up of membranes of the DMS.

Ultrastructural cytochemical studies of the oxidation of 3,3 -diaminobenzidine have demonstrated a platelet peroxidase (PPO) in the endoplasmic reticulum and perinuclear space but not in the Golgi apparatus of megakaryoblasts and megakaryocytes and in the dense bodies and dense tubular system of platelets (85). A few small rounded cells present in normal marrow also have PPO activity in the endoplasmic reticulum and perinuclear space and have been identified as promegakaryoblasts (86). Platelet peroxidase appears to be distinct from myeloperoxidase.

Some normal megakaryocytes display the phenomenon of emperipolesis (87,88). This term is used to describe the movement of one cell type within the cytoplasm of another. The cytoplasm of an affected megakaryocyte may contain one or more cells of a number of types, including neutrophil and eosinophil granulocytes and their precursors, lymphocytes, erythroblasts, and red cells (Figure 33.50). The physiologic relevance of megakaryocyte emperipolesis is uncertain; one suggestion has been that certain marrow cells may enter the circulation via the processes of megakaryocyte cytoplasm that protrude into marrow sinusoids.

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Nonactivated platelets are biconvex and have a smooth surface. Their shape is maintained by an equatorial bundle of microtubules situated below the cell membrane, as well as by microfilaments found between various organelles. Other structures found in the cytoplasm include various types of granules, mitochondria, a surface-connected canalicular system, the dense tubular system, and many glycogen particles, which may occur singly or in clumps (Figure 33.52).

Four types of cytoplasmic granule are recognized, namely, the granules, granules (lysosomal granules), granules, and peroxisomes (75,89,90). The and granules are moderately electron dense and can be distinguished from each other only by ultrastructural cytochemistry; for example, granules have acid phosphatase activity and granules do not. Substances present in granules include -thromboglobulin, platelet factor 4, platelet-derived growth factor, fibrinogen, fibronectin, von Willebrand factor, and thrombospondin. In addition to acid phosphatase, the granules contain -glucuronidase and arylsulfatase. The granules (dense granules) are smaller and much more electron dense than granules and often have a peripheral electron-lucent zone, which gives them a bull's-eye appearance. They contain serotonin, calcium, and the storage pool of ADP and ATP. The peroxisomes are smaller than the and granules; they are moderately electron dense and contain catalase.

The surface-connected canalicular system is an extensive system of electron-lucent intracytoplasmic canaliculi and saccules that open to the exterior at multiple sites on the cell membrane. This canalicular system provides a large surface through which various substances, including granule contents, could be discharged extracellularly. The channels of the dense tubular system are shorter and narrower than those of the surface-connected canalicular system and contain material with an electron density similar to that of the cytoplasm. The dense tubular system contains platelet peroxidase and seems to be derived from the endoplasmic reticulum of megakaryocytes. It is an important site of synthesis of thromboxane A₂, which is involved in the release of granule contents. It is also rich in calcium and may regulate various calcium-dependent reversible reactions such as the activation of actomyosin and the polymerization of tubulin.

Lymphocytes and Plasma Cells

All lymphocytes are eventually derived from the lymphoid stem cells present in the marrow, which are in turn derived from the pluripotent hematopoietic stem cells. The lymphoid stem cells generate both B-cell progenitors and T-cell progenitors. The former mature through a number of antigen-independent intermediate stages into B cells; this maturation occurs within the microenvironment of the marrow. The newly formed B cells travel via the blood into the B-cell zones of peripheral lymphoid tissue. Either the lymphoid stem cells or early T-cell progenitors migrate from the marrow through the blood into the thymus. Here, these cells undergo antigen-independent maturation into T cells, and those T cells that recognize self are deleted. The mature T cells then travel through the blood into the T-cell zones of the peripheral lymphoid organs. The mature B and T lymphocytes that enter the peripheral lymphoid tissue are triggered into division when they react with specific antigen in the presence of appropriate accessory cells. Their progeny develop into effector cells or memory cells. In the case of B cells, the effector cell is an antibody-secreting plasma cell. Antigen-dependent proliferation of B cells occurs in normal marrow and results in the presence of plasma cells in this tissue.

Immunohistochemical studies show that the ratio of T cells to B cells in normal adult marrow is around 3:1. The light and electron microscopic appearances of bone marrow lymphocytes are indistinguishable from those of other lymphocytes in the body. Some T and B lymphocytes have fine or coarse PAS-positive granules arranged in one to four (usually one or two) rings around the nucleus, and occasional cells have large clumps of PAS-positive material. Lymphocytes are peroxidase negative and -naphthol AS-D chloroacetate esterase negative, and over 99% of cells are alkaline phosphatase negative. Some lymphocytes

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Plasma Cells

Plasma cells seen in smears of normal bone marrow vary considerably in size and appearance. Most are 14 to 20 m in diameter and have deep blue cytoplasm. The cytoplasm has a pale paranuclear area corresponding to the Golgi apparatus and may contain one or more vacuoles (Figure 33.53A B). The nucleus is small relative to the volume of cytoplasm, contains moderate quantities of condensed chromatin, and is eccentrically located. Although most plasma cells are uninucleate, a few are binucleate or multinucleate. Some normal plasma cells have other features. For example, occasional cells may contain one or a few large rounded acidophilic, PAS-positive cytoplasmic inclusions (Russell bodies) or several smaller slightly basophilic rounded inclusions (Mott cells, grape cells, or morular cells). Some plasma cells have many pleomorphic cytoplasmic inclusions and, consequently, appear reticulated (Figure 33.53C). Others have eosinophilic cytoplasm, usually at the periphery, but sometimes in the entire cell (flaming cell); when the eosinophilia is confined to the periphery, it contrasts markedly with the intense basophilia of the rest of the cytoplasm. Occasional plasma cells have azurophilic rods that resemble Auer rods present in acute myeloid leukemia but that are PAS, Sudan black, and peroxidase negative. Plasma cells show strong acid phosphatase activity, particularly around the nucleus and over the Golgi zone. They do not stain for -naphthol AS-D chloroacetate esterase.

The electron microscope shows that the eccentric rounded nucleus of a plasma cell contains a variable quantity of condensed chromatin (Figures 33.54,33.55A) and a well-developed nucleolus (Figure 33.55A). The presence of moderately large clumps of nuclear membrane associated condensed chromatin gives the nuclei of mature plasma cells a cartwheel or clock face appearance in histologic sections (but not in marrow smears). The cytoplasm contains numerous long flattened sacs of RER that are arranged either parallel to each other (Figure 33.56), concentrically, or spirally; the sacs are distended to varying extents with a granular, moderately electron-dense material, consisting mostly of immunoglobulin. The cytoplasm also contains mitochondria, a large Golgi apparatus situated immediately adjacent to the nuclear membrane (Figure 33.54), and a few small or medium-sized membrane-bound electron-dense granules. The latter are often found near the Golgi complex, contain acid phosphatase, and appear to be primary lysosomes. Occasional cells contain larger cytoplasmic inclusions that vary markedly in size, electron density, and shape

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Hematopoietic Cells: Characteristics in Histologic Sections

In H&E-stained sections of formalin-fixed paraffin-embedded trephine biopsies, insufficient cytoplasmic basophilia and nuclear detail is seen to enable reliable distinction between myeloblasts, neutrophil promyelocytes, neutrophil myelocytes, and early erythroblasts. However, neutrophil metamyelocytes and band cells can be recognized by their C- or U-shaped nuclei and neutrophil granulocytes by the presence of two or more darkly staining nuclear lobes or segments lying close together (Figure 33.57). In histologic sections, the fine chromatin strands that join the nuclear lobes of granulocytes usually are not seen. The cytoplasm of neutrophil myelocytes and metamyelocytes stains pale pink and that of neutrophil granulocytes a very pale pink. The granules contained within cells of the neutrophil series stain poorly and are usually difficult to see. Neutrophil promyelocytes and myelocytes can be reliably distinguished from immature cells belonging to other cell lineages by immunohistochemical staining of neutrophil series specific antigens such as neutrophil elastase (Table 33.1). In sections of paraffin-embedded marrow fragments, the neutrophil promyelocytes/myelocytes, metamyelocytes, and granulocytes are stained by Leder's stain for chloroacetate esterase (Figure 33.58A). These cells are also stained, both in sections of marrow fragments and trephine biopsy cores, by the PAS reaction (Figure 33.58B). Eosinophil myelocytes, metamyelocytes, and myelocytes can be readily recognized by their red-orange cytoplasm, resulting from the presence of large eosinophilic granules (Figure 33.57). Because basophil granules are water soluble, their contents become extracted during routine fixation for histologic studies. Consequently, basophil granulocytes cannot be seen in histologic sections processed in the usual way.

Erythroblasts of varying degrees of maturity are found in distinctive clumps. Pronormoblasts and basophilic normoblasts are large cells with rounded nuclei containing nucleoli. They only show slight cytoplasmic basophilia when stained by H&E and thus resemble early granulocyte precursors; their identification is therefore based largely on their association with groups of more mature erythroblasts. The late erythroblasts contain rounded heavily stained nuclei showing little structural detail and have moderate quantities of poorly staining cytoplasm, usually with a distinct cytoplasmic membrane. They may show clear halos around the nucleus as a consequence of the shrinkage of the cytoplasm (Figure 33.59). Lymphocytes do not show this artifact. Erythroblasts can be reliably identified by immunohistochemical staining of glycophorins A and C and of hemoglobin A (Table 33.1, Figure 33.60).

The Giemsa stain is superior to H&E for identifying myeloblasts and promyelocytes as well as pronormoblasts and basophilic normoblasts, staining their cytoplasm blue. However, it is still not possible to reliably distinguish myeloblasts from promyelocytes. In Giemsa-stained sections, pronormoblasts have more basophilic cytoplasm than do other blasts and promyelocytes (Figure 33.61).

Lymphocytes may be difficult to distinguish from late erythroblasts in histologic sections of paraffin-embedded trephine biopsy cores except when present in lymphoid nodules. They have a narrow rim of slightly basophilic (Giemsa) or poorly staining (H&E) cytoplasm, indistinct cytoplasmic margins, and a clumped nuclear chromatin pattern. The nuclei of lymphocytes are less perfectly rounded and more variable in size and shape and show more structural detail than those of late erythroblasts. In H&E-stained sections, plasma cells can be identified by the presence of slightly or moderately basophilic cytoplasm, an eccentric nucleus with a cartwheel or clock face chromatin pattern, and a pale paranuclear zone corresponding to the Golgi apparatus. In Giemsa-stained sections, the cytoplasm of many plasma cells stains a deep blue and, consequently, the pale Golgi zone is especially prominent.

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Megakaryocytes are readily recognized by their large size, light or dark pink cytoplasm, and lobulated nucleus in sections stained either with hematoxylin and eosin or Giemsa (Figure 33.62). In sections of normal bone marrow they are present in clusters of two to five cells and are usually not found in a paratrabecular position. Small numbers of bare megakaryocyte nuclei, with convolutions and a considerable quantity of condensed chromatin, also are seen.

B and T lymphocytes, plasma cells (Figure 33.22A B), and megakaryoctyes (Figure 33.22D) can be identified immunohistochemically, and megakaryoblasts can be reliably identified only in this way (Table 33.1). Using the monoclonal antibody Y2/51 which is directed against Gp IIIa, the mean value for the total number of megakaryocytes and megakaryoblasts in 15 normal subjects was 24/mm² (range: 14 38) and for megakaryoblasts alone it was 2.8/mm² (range: 1.2 4.9) (91).

As has already been mentioned, much more cytologic detail and especially nuclear detail can be seen in semithin sections of undecalcified plastic-embedded trephine cores (Figure 33.63) than in convention sections of decalcified paraffin-embedded cores (Figure 33.62A).

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Cellularity of the Marrow

The term marrow cellularity usually is defined as the proportion of the area of a histologic section excluding bone occupied by hematopoietic cells (by cells other than fat cells). Cellularity is usually assessed by point counting using an eyepiece with a graticule (histomorphometry) or, more accurately, by computerized image analysis (92). The shrinkage of tissue subjected to decalcification and paraffin embedding results in the cellularity of paraffin-embedded sections being about 5% lower than in plastic-embedded sections (93).

In healthy subjects, cellularity varies with age (53,54). In neonates, there are very few fat cells in the marrow, and the cellularity approaches 100%. Cellularity decreases steadily in the first three decades and stabilizes at 30 to 70% between the ages of 30 and 70 years. During the eighth decade of life, cellularity decreases further and may be less than 20%; this reduction is largely caused by a reduction in bone volume and a consequent increase in the volume of the marrow cavities.

In assessing cellularity, it should be noted that cellularity varies markedly from one intertrabecular space to the next in a single biopsy specimen so that a reliable estimate requires the examination of at least five such spaces (i.e., a biopsy core of greater than 2 cm). Furthermore, the immediate subcortical marrow of the ilium is frequently less cellular than deeper marrow. A study of postmortem biopsy samples from 100 normal subjects who died suddenly without evidence of bone or marrow disease showed only slight differences in the cellularity at different hematopoietic sites. The percentage cellularity ( SD) in biopsies from the anterior iliac crest, posterior iliac crest, lumbar vertebrae, and sternum were, 60 6, 62 7, 64 7, and 61 8, respectively (94).

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Marrow Differential Count

During the first day of life, the erythroblasts account for 18.5 to 65% (mean: 40%) of the nucleated cells in a marrow smear. Over the next 8 to 10 days, this figure decreases progressively to 0 to 20.5% (mean: 8%). After a period of erythroblastopenia lasting about three weeks, the percentage of erythroblasts increases again, reaching values of 6.5 to 31.5% (mean: 16%) at the age of 3 months (95). These changes are caused by an increase in arterial oxygen saturation soon after birth and the consequent suppression of erythropoietin production. Erythropoietin production increases again 6 to 13 weeks later when the hemoglobin concentration in the blood decreases to about 11 g/dL. The proportion of granulocytes and their precursors ranges between 20 and 73% (mean: 46%) of the nucleated marrow cells on the first day of life (95), increases during the next three weeks, and then decreases again to reach a stable value of about 55% after the second month. The average value for the proportion of lymphocytes in the marrow increases from 12% during the first two days of life, to 33% at seven to ten days, and 47% at one month. The lymphocyte percentage then remains stable until the end of the first year, after which it decreases slowly to 19% at 4 to 4.5 years, which is only slightly higher than the adult value of 15% (95,96,97,98). Plasma cells are infrequent in the neonate, accounting for up to 0.4% (mean: 0.016%) of nucleated marrow cells (99). They gradually increase in number to reach a mean value of 0.386% at the age of 12 to 15 years (5000 cell differential count). When 300 to 500 consecutive marrow cells are assessed in each case, the prevalence of plasma cells in healthy adults is 0.4 to 4.0%.

The differential count on 2000 consecutive nucleated cells in bone marrow smears from normal adults is given in Table 33.2 (100). The mean and range for the myeloid/erythroid ratio in healthy adults are 3.1 and 2.0:8.3, respectively (101).

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