26 - Vermiform Appendix
Editors: Mills, Stacey E.
Title: Histology for Pathologists, 3rd Edition
Copyright 2007 Lippincott Williams & Wilkins
> Table of Contents > VIII - Hematopoietic System > 33 - Bone Marrow
function show_scrollbar() {}
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
P.800
organization. However, this organization is labile, altering rapidly in response to many stimuli.
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.
Table 33.1 Some Monoclonal and Polyclonal Antibodies that can be used in Immunohistochemical Studies on Sections of Decalcified, Paraffin-Embedded Trephine Biopsies of the Marrow | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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).
P.801
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
P.802
generate new stem cells and thus to maintain their own number (self-renewal). In humans, the existence of pluripotent hematopoietic stem cells with both the above properties has been demonstrated by the success of bone marrow transplantation. The marrow also contains primitive cells that form cobblestone foci of myelopoiesis under stromal cells in long-term marrow cultures (limited to 8 20 weeks); these do not have the capacity for extensive self-renewal or for long-term lymphopoiesis and are therefore considered to be more differentiated than stem cells. The pluripotent stem cells (25,26,27) give rise to lymphoid stem cells and multipotent myeloid stem cells. They may also give rise to endothelial cells (28). The lymphoid stem cells mature into all types of lymphocytes. The myeloid stem cells mature into neutrophil, eosinophil, and basophil granulocytes, monocytes, erythrocytes, platelets, mast cells, and osteoclasts. The immediate progeny of the lymphoid and myeloid stem cells are usually termed hematopoietic progenitor cells. The myeloid progenitor cells are committed to one, two, or a few hematopoietic differentiation pathways (i.e., are unipotent, bipotent, or oligopotent) and have only a limited capacity for self-renewal. The most immature of the myeloid progenitor cells are oligopotent. Such cells undergo a progressive restriction of their differentiation potential such that the most mature progenitor cells are committed to only a single line of differentiation. Hematopoietic progenitor cells have been identified and characterized by their ability to form colonies containing cells of one or more hematopoietic lineages in vitro and are therefore called colony-forming units (CFUs) or colony-forming cells (CFC). These generate colonies containing a mixture of granulocytes, erythroblasts, macrophages, and megakaryocytes and are, therefore, termed CFU-GEMM. There is some indirect evidence for the presence of tripotent hematopoietic cells (CFU-E mega baso) that give rise to erythroblasts, megakaryocytes, and basophil granulocytes. Bipotent hematopoietic progenitor cells that give rise to colonies containing granulocytes and macrophages are termed CFU-GM. There are also bipotent progenitor cells generating colonies containing a mixture of erythroblasts and megakaryocytes (CFU-E mega). The unipotent progenitor cells that give rise to neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, macrophages, erythroblasts, and megakaryocytes are described as CFU-G, CFU-eo, CFU-baso, CFU-M, CFU-E, and CFU-mega, respectively. These develop into the most immature of the morphologically recognizable blood cell precursors in the marrow. Thus, CFU-G develop into myeloblasts, CFU-eo into eosinophil promyelocytes, CFU-baso into basophil promyelocytes, CFU-M into monoblasts, CFU-E into pronormoblasts, and CFU-mega into megakaryoblasts. The stem cells and progenitor cells are found in both the blood and the marrow but cannot be identified on morphologic criteria. The characteristics of the various types of morphologically recognizable hematopoietic cell found in the marrow are described later in this chapter. The relationships between the different categories of cell involved in hematopoiesis are illustrated in Figure 33.1.
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).
P.803
 |
Figure 33.1 Model of hematopoiesis showing the relationships between the various types of stem cell, progenitor cell, and morphologically recognizable precursor cell. (BFU, E, erythroid burst-forming units; CFU, colony-forming units; E, erythroblasts; GM, granulocytes and macrophages; eo, eosinophil granulocytes; baso, basophil granulocytes; mega, megakaryocytes; G, neutrophil granulocytes; M, macrophages) |
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.
P.804
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).
 |
Figure 33.2 Semithin section of a plastic-embedded chorionic villus biopsy sample obtained at 7 weeks of gestation, showing a blood vessel containing nucleated embryonic red cells (toluidine blue). |
 |
Figure 33.3 Fetal liver tissue obtained postmortem showing erythropoietic activity. The erythroblasts are found extravascularly, both within the hepatic cords and between the cords and the sinusoidal endothelial cells. The brown material within hepatocytes is formalin pigment, a common postmortem fixation artifact. (H&E.) |
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
P.805
erythroblasts) remains constant at about 1:4 after 6.5 months of gestation (40).
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
P.806
hematopoietic factors derived from the bone or endosteal cells.
 |
Figure 33.4 Electron micrograph of part of the wall of a sinusoid from normal bone marrow. There are three tight junctions (small arrows) at the area of contact between two adjacent endothelial cells. Several pinocytotic vesicles (large arrow) are present both at the luminal and abluminal surface of one of the endothelial cells, and a single pinocytotic vesicle is present at the outer surface of the adventitial cell. |
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
P.807
near the endosteum; such fibers are continuous with the fibers in the parenchyma. Other extracellular matrix components produced by stromal cells include fibronectin, vascular cell adhesion molecule (VCAM)-1, vitronectin, thrombospondin, and proteoglycans such as heparan sulphate and chondroitin sulphate.
 |
Figure 33.5 Section of a decalcified, paraffin-embedded trephine biopsy core from a hematologically normal adult, showing a scant network of fine reticulin fibers. The upper right-hand quadrant of the photomicrograph shows a circular arrangement of fibers associated with a blood vessel. (Silver impregnation of reticulin.) |
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.
 |
Figure 33.6 Group of osteoblasts from a May-Gr nwald-Giemsa (MGG) stained smear of normal bone marrow. |
 |
Figure 33.7 A multinucleate osteoclast from an MGG-stained smear of normal bone marrow. |
 |
Figure 33.8 Section of a decalcified, paraffin-embedded trephine biopsy core from a hematologically normal adult. About 70% of the area of marrow tissue in this photomicrograph is occupied by fat cells. There may be a substantial variation in cellularity in different parts of the same section. (H&E.) |
P.808
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).
 |
Figure 33.9 Semithin section of an undecalcified, plastic-embedded trephine biopsy core from a hematologically normal adult. A wide sinusoid containing red cells is seen passing vertically between some fat cells. (H&E.) |
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
P.809
phagocytosed material (Figure 33.11A). Macrophages are derived from monocytes and, therefore, eventually from the hematopoietic stem cells.
 |
Figure 33.10 Immunohistochemical demonstration of macrophages in a section of a paraffin-embedded trephine biopsy core from a hematologically normal subject. The section was reacted with the monoclonal antibody PG-M1 (against CD68) and the reaction visualized using an immunoperoxidase technique. |
 |
Figure 33.11 Macrophages from normal bone marrow smears. A. Macrophage containing a black extruded erythroblast nucleus and several intracytoplasmic inclusions of various shapes, sizes, and staining characteristics. The large pale rounded inclusions may represent degraded red cells (MGG). B. Macrophage containing several PAS-positive cytoplasmic granules, together with a PAS-negative late erythroblast and several PAS-positive neutrophil myelocytes and granulocytes. C: Macrophage showing strong -naphthyl acetate esterase activity, surrounded by six unreactive erythroblasts. The diazonium salt of fast blue BB was used as the capture agent. |
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)
P.810
and acid phosphatase. They do not stain for -naphthol AS-D chloroacetate esterase activity (57), and most do not stain with Sudan black. Some macrophages appear to stain positively for alkaline phosphatase activity.
 |
Figure 33.12 Section of a paraffin-embedded normal marrow fragment (clot section). The macrophage in the center shows blue hemosiderin-containing intracytoplasmic granules and a diffuse bluish coloration of the cytoplasm (Perls' acid ferrocyanide reaction). |
 |
Figure 33.13 Marrow fragment from a normal marrow smear stained by Perls' acid ferrocyanide reaction. The dark blue granular material represents hemosiderin within macrophages. |
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
P.811
(e.g., c-kit ligand or stem cell factor, M-CSF, IL-1, and G-CSF) and are thus involved in short-range regulation of lymphopoiesis and myelopoiesis. They presumably also are involved in antigen processing.
 |
Figure 33.14 Electron micrograph of three erythroblasts from a normal marrow showing fine processes of macrophage cytoplasm extending between the cells. |
 |
Figure 33.15 Electron micrograph of a sinusoid from a normal bone marrow. A process of macrophage cytoplasm is seen protruding through the lining endothelial cell into the sinusoidal lumen. Serial sectioning of this sinusoid showed that the mass of macrophage cytoplasm occupying the right-hand side of the sinusoidal lumen connected transendothelially with a second extrasinusoidal cytoplasmic process. Both processes arose from the same macrophage. |
 |
Figure 33.16 Electron micrograph of a macrophage lying next to an early polychromatic erythroblast in a normal bone marrow. The nucleus of the macrophage is irregular in outline, and its cytoplasm contains several inclusions and vacuoles. Some of the inclusions are ultrastructurally complex and probably represent secondary lysosomes. There are some reticulin fibers (arrow) near the macrophage. |
 |
Figure 33.17 Electron micrograph of a macrophage from normal bone marrow. The cytoplasm contains two phagocytosed neutrophils and a large number of other inclusions of varying size, shape, and appearance. |
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).
 |
Figure 33.18 Electron micrographs of two nonphagocytic reticular cells from normal bone marrow. The nuclear outline of one of these cells (A) shows several deep clefts and that of the other (B) is less irregular. |
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.
 |
Figure 33.19 Two mast cells, one rounded (A) and one elongated (B) from MGG-stained smears of bone marrow. |
P.812
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
P.813
reticulin content is normal for marrow or only slightly increased. The remaining 80% are rounded or oval, well circumscribed, and compact, and they have a follicular structure with blood vessels at their center and some plasma cells and mast cells toward their periphery. Well-developed germinal centers are seen in about 5% of the sections. Lymphocytes extend between surrounding fat cells, and the entire nodule may be surrounded by eosinophils. The reticulin content of such a lymphoid nodule is clearly increased (Figure 33.21B). Immunohistochemical studies indicate that the lymphocytes within such nodules are of both T and B phenotypes (mainly T).
 |
Figure 33.20 Electron micrographs of mast cells from normal bone marrow. A. The cytoplasm is packed with characteristic granules and contains four lipid droplets. B. Granules from a mast cell at high magnification showing parallel lamellae. |
 |
Figure 33.21 Lymphoid nodule in a paraffin-embedded trephine biopsy core from a woman without any evidence of a lymphoproliferative disorder. A. Section stained with hematoxylin and eosin showing a nodule (top right) incorporating fat cells at its periphery. B. Parallel section stained by a silver impregnation technique, showing increased reticulin in the nodule (photographed at higher magnification than A). |
P.814
 |
Figure 33.22 Immunohistochemical studies on sections of paraffin-embedded trephine biopsy cores. The reactions were visualized using an immunoalkaline phosphatase method. A. Section reacted with anti-Ig light chains, showing plasma cells aligned along a blood vessel. B. Section reacted with anti-Ig light chains showing a cluster of plasma cells. There are fewer positive plasma cells with anti- chain than anti- chain antibody. C. Section reacted with antimyeloperoxidase antibody showing positively-stained promyelocytes and more mature cells of the neutrophil granulocyte series. Note the presence of stained cells along the endosteal surface of the bone trabeculum. D. Section reacted with antibody against factor VIII related antigen showing positively stained megakaryocytes and linearly arranged endothelial cells. One of the megakaryocytes is closely apposed to the blood vessel. (Courtesy of Dr. Alex Rice, Department of Histopathology, St. Mary's Hospital, London). |
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
P.815
classes through which myeloblasts mature into circulating neutrophil granulocytes are termed neutrophil promyelocytes, neutrophil myelocytes, neutrophil metamyelocytes, juvenile neutrophils, and marrow neutrophil granulocytes (Figures 33.23,33.24). Cell division occurs in myeloblasts, promyelocytes, and myelocytes but not in more mature cells.
 |
Figure 33.23 Neutrophil precursors from an MGG-stained normal marrow smear. A. A myeloblast, an early promyelocyte, and a late promyelocyte/early myelocyte. B. A promyelocyte and a neutrophil granulocyte. |
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.
 |
Figure 33.24 Two neutrophil myelocytes (one large and one small), a neutrophil metamyelocyte, and a juvenile neutrophil (stab form) from a normal marrow smear. |
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
P.816
more mature cells of the neutrophil series stain positively with the PAS reagent, with Sudan black, and with reactions for peroxidase and -naphthol AS-D chloroacetate esterase activity. A granular staining pattern is produced with all these cytochemical reactions (Figure 33.25). The intensity of staining increases in cell classes of increasing maturity with the PAS reaction and, to a lesser extent, with Sudan black. Promyelocytes and neutrophil myelocytes, but not neutrophil granulocytes, stain for -naphthyl acetate esterase activity and, more weakly, for -naphthyl butyrate esterase activity. Acid phosphatase activity is present in cells at and after the promyelocyte stage; this activity is strongest in the immature cells and weak in neutrophil granulocytes. A few neutrophil metamyelocytes stain weakly for alkaline phosphatase activity, and segmented neutrophil granulocytes stain with a variable intensity (weak to strong) (24,69,70,71). Immunocytochemical studies indicate that both lysozyme (muramidase) and elastase are present in promyelocytes and all of the more mature cells of the neutrophil series and that lactoferrin is present in neutrophil myelocytes, metamyelocytes, and granulocytes.
 |
Figure 33.25 Cytochemical reactions of neutrophil precursors and neutrophil granulocytes. A. Faint PAS positivity in neutrophil myelocytes and stronger positivity in neutrophil granulocytes. The three erythroblasts are PAS negative. B. Sudan black positivity in two neutrophil myelocytes, one eosinophil myelocyte, a neutrophil metamyelocyte, and a neutrophil granulocyte. The lymphocytes and erythroblasts are sudanophobic. C. Strong peroxidase positivity in neutrophil myelocytes and granulocytes; p-phenylene diamine and catechol were used as the substrate. D. Alpha-naphthol AS-D chloroacetate esterase positivity in three neutrophil myelocytes and a neutrophil granulocyte. The two erythroblasts have not stained. The diazonium salt of fast violet-red LB was used as the capture agent. |
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
P.817
granulocyte stages. The cytoplasm of a promyelocyte characteristically contains a variable number of immature and mature primary granules. Mature primary granules are elliptical, measure 0.5 to 1.0 m in their long axis, are electron dense, and contain peroxidase, lysozyme, elastase, 1 antitrypsin, and sulphated mucosubstances. Some have a core with a linear periodic substructure. Ultrastructurally different granules, the secondary granules, are found in addition to primary granules at the neutrophil myelocyte stage (Figures 33.26,33.27). Secondary granules are larger and less electron dense than primary granules, have rounded outlines, tend to undergo a variable degree of extraction, and are only peroxidase positive if a high concentration of diaminobenzidine is used at alkaline pH. They contain lysozyme and vitamin B12 binding protein. Another variety of granule, known as tertiary granules, is present at and after the metamyelocyte stage. These granules are small (0.2 0.5 m in their long axis), pleomorphic (including rounded, elongated, or dumbbell-shaped forms), and peroxidase negative. Their electron density is usually between that of primary and secondary granules (Figure 33.28). Other electron microscopic cytochemical studies have shown that acid phosphatase is present in primary granules but not in secondary or tertiary granules. The above data on the distribution of peroxidase and acid phosphatase suggest that secondary and tertiary granules do not arise from the modification of primary granules but are synthesized de novo at the myelocyte and metamyelocyte stages, respectively (72). Immunoelectron microscopy has demonstrated that lactoferrin is only found in some of the granules at and after the neutrophil myelocyte stage. The alkaline phosphatase activity in neutrophil granulocytes is present within small membrane-bound intracytoplasmic vesicles called phosphosomes.
 |
Figure 33.26 Electron micrograph of an immature neutrophil myelocyte from normal bone marrow. The nucleus contains a prominent nucleolus and a small quantity of nuclear membrane associated condensed chromatin. The cytoplasm contains several strands of endoplasmic reticulum, a prominent paranuclear Golgi apparatus, and two ultrastructurally distinct types of granules. |
 |
Figure 33.27 Part of the cytoplasm of the cell in Figure 33.26 at higher magnification. Two types of granules can be clearly recognized. These are (a) rounded or elliptical, very electron-dense primary granules (formed at the promyelocyte stage) and (b) larger, rounded, less electron-dense secondary granules (formed at the myelocyte stage). |
The primary granules observed with the electron microscope correspond to the azurophilic granules seen in Romanowsky-stained smears, and the secondary and tertiary
P.818
granules correspond to the neutrophilic or specific granules. Although primary granules are present in all granule-containing cells of the neutrophil series, they lose their azurophilic property and are therefore not detectable by light microscopy at and after the metamyelocyte stage.
 |
Figure 33.28 Electron micrograph of a neutrophil granulocyte from a normal bone marrow. In addition to some primary and secondary granules, the cytoplasm contains several small pleomorphic tertiary granules. |
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.
 |
Figure 33.29 Cells from an MGG-stained normal bone marrow smear. The cell types shown are, from left to right, an eosinophil myelocyte, a plasma cell, a neutrophil granulocyte, and a lymphocyte. |
 |
Figure 33.30 Basophil granulocyte from an MGG-stained normal marrow smear. |
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
P.819
secondary granules contain a central electron-dense crystalloid inclusion consisting largely of polymerized major basic protein. It is generally held that the primary granules mature into secondary granules. Early eosinophil promyelocytes contain only primary granules, but more mature promyelocytes contain many primary and a few secondary granules. Eosinophil myelocytes contain some primary and several secondary granules (Figure 33.31). By contrast, the majority of the granules in eosinophil metamyelocytes and granulocytes are secondary granules (Figure 33.32). The primary granules of eosinophil promyelocytes are larger and more rounded than the primary granules of neutrophil promyelocytes and promonocytes.
 |
Figure 33.31 Electron micrograph of part of the cytoplasm of an early eosinophil myelocyte. A centriole surrounded by well-developed Golgi saccules, several strands of rough endoplasmic reticulum, and a number of large granules are seen. Some of the granules are homogeneously electron dense (primary granules), but others have a central crystalloid (secondary granules). |
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.
 |
Figure 33.32 Electron micrograph of an eosinophil granulocyte from normal bone marrow. The majority of the cytoplasmic granules are crystalloid-containing secondary granules. Note that the uppermost granule is unusual in that its crystalloid stains more lightly than the surrounding granule matrix. |
Monoblasts are similar in appearance to myeloblasts except that their nuclei may be slightly indented or lobulated.
P.820
They have small quantities of agranular deeply basophilic cytoplasm and can only be reliably distinguished from other types of blast cells by cytochemical and other special techniques (Figure 33.35). Promonocytes are larger, have a lower nucleus-to-cytoplasm ratio, and contain less basophilic cytoplasm than monoblasts; their cytoplasm contains a few azurophilic granules. Promonocytes usually have a large, rounded, cleft or lobulated nucleus with the chromatin appearing as a fine network. Nucleoli may or may not be visible.
 |
Figure 33.33 Electron micrograph of a basophil granulocyte from a normal bone marrow. The granules have been markedly extracted during processing, but the characteristic closely packed rounded particles can still be recognized in several of the granules. |
 |
Figure 33.34 Electron micrograph illustrating the particulate ultrastructure of basophil granules at high magnification. |
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.
 |
Figure 33.35 A cell with strong -naphthyl acetate esterase activity from a normal marrow smear (the diazonium salt of fast blue BB was used as the capture agent). This cell has a slightly convoluted nucleus and relatively little cytoplasm and is most probably a monoblast or early promonocyte. |
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
P.821
monocytes. All the promonocyte granules and some of the monocyte granules are peroxidase positive.
 |
Figure 33.36 Electron micrograph of an immature monocyte from normal bone marrow. The cytoplasm contains many small mature granules and a few immature granules (see Figure 33.37). Several short cytoplasmic processes can be seen at the periphery of the cell. |
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).
 |
Figure 33.37 A higher-power view of part of the cytoplasm of the cell in Figure 33.36 showing a few immature-looking granules with an electron-dense central zone and an electron-lucent peripheral zone. There are also some small, uniformly electron-dense mature granules. |
 |
Figure 33.38 Red cell precursors from a normal bone marrow smear (A C) and a reticulocyte from normal peripheral blood (D). A. Pronormoblast. B. Two early polychromatic normoblasts and two late polychromatic normoblasts. C. A sideroblast showing a fine, barely visible blue siderotic granule. (A and B, MGG stain; C, Perls' acid ferrocyanide reaction; D, supravital staining with brilliant cresyl blue.) |
P.822
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
P.823
P.824
vacuoles are found in 22% and slight to substantial degrees of myelinization of the nuclear membrane in 12% of erythroblast profiles (79). Other data shown by electron microscopic studies of the erythron are that (a) part of the cell's cytoplasmic membrane and a narrow rim of hemoglobin-containing cytoplasm completely surrounds the extruded erythroblast nucleus (Figure 33.44); (b) the marrow reticulocytes enter the sinusoids by passing through, rather than between, endothelial cells (Figure 33.45); and (c) whereas reticulocytes contain ribosomes and mitochondria, mature red cells do not.
 |
Figure 33.39 Part of an early polychromatic erythroblast showing a rhopheocytotic surface invagination with a few adherent ferritin molecules. A rhopheocytotic vesicle containing several ferritin molecules is closely apposed to the surface invagination. A narrow process of ferritin-containing macrophage cytoplasm is present between the erythroblast displaying rhopheocytosis and the adjacent cell. |
 |
Figure 33.40 Electron micrograph of a pronormoblast from normal bone marrow. The nucleus contains very small quantities of condensed chromatin and has a prominent nucleolus. The cytoplasm is relatively electron lucent and rich in polyribosomes. |
 |
Figure 33.41 Electron micrograph of a group of six erythroblasts at various stages of maturation. Note that maturation is associated with an increase in the electron density of the cytoplasm. The lowermost cell is a late erythroblast about to extrude its nucleus. |
 |
Figure 33.42 Electron micrograph of part of the cytoplasm of a polychromatic erythroblast from normal bone marrow. The cytoplasm shows a membrane-bound accumulation of ferritin and hemosiderin (siderosome) (arrow) and a few ferritin-containing rhopheocytotic vesicles. |
 |
Figure 33.43 Part of a polychromatic erythroblast from a normal marrow showing a membrane-bound siderosome that is much more densely packed with ferritin and hemosiderin molecules than the siderosome in Figure 33.42. |
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
P.825
proliferation or maturation in the affected cells. In many congenital or acquired disorders characterized by grossly disordered erythropoiesis, the proportion of erythroblasts showing these dyserythropoietic changes is increased, and some erythroblasts show various dyserythropoietic changes not seen in normal marrow (43). The latter include nonspecific abnormalities, such as large autophagic vacuoles and extensive intranuclear clefts, as well as abnormalities that are specific for certain diseases or groups of diseases.
 |
Figure 33.44 Electron micrograph of an extruded erythroblast nucleus. Note that the nucleus is surrounded by a rim of hemoglobin-containing cytoplasm and lies in close contact with processes of macrophage cytoplasm. |
 |
Figure 33.45 Electron micrograph illustrating an uncommon mechanism of formation of a reticulocyte. Whereas nuclear expulsion often occurs extravascularly and the resulting reticulocytes then enter a sinusoid, the cytoplasm of the late erythroblast shown has passed through the endothelial cell of the sinusoid before nuclear expulsion. The nucleus of this erythroblast has not passed through the narrow passage in the endothelial cell and presumably will be severed from the rest of the cell and phagocytosed by the macrophage (arrow) lying in contact with it. Thus, in this erythroblast, nuclear expulsion appears to occur during entry of the future reticulocyte into the sinusoid. |
 |
Figure 33.46 Morphologic evidence of dyserythropoiesis in bone marrow smears from healthy volunteers. A. Intererythroblastic cytoplasmic bridge. B. Large Howell-Jolly body in an early polychromatic erythroblast. C. Two smaller Howell-Jolly bodies in a late polychromatic erythroblast. D. Karyorrhexis in a late polychromatic erythroblast. |
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
P.826
fairly tightly packed together before the shedding of platelets. The nuclear chromatin has a coarse-grained appearance. Platelets are formed by the fragmentation of cytoplasmic processes of the mature granular megakaryocytes. When platelet formation is completed, a bare nucleus remains.
 |
Figure 33.47 Granular megakaryocyte from an MGG-stained normal bone marrow smear. |
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.
 |
Figure 33.48 Megakaryocyte from a normal bone marrow smear showing large quantities of PAS-positive material. |
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
P.827
membrane-lined vesicles representing the beginning of the demarcation membrane system (DMS). The cytoplasm also contains a well-developed Golgi apparatus within a deep nuclear indentation. A few immature granules and a few lysosomal vesicles containing acid phosphatase and arylsulfatase are present near the Golgi apparatus. The maturation of megakaryoblasts into promegakaryocytes and granular megakaryocytes (Figure 33.50) is accompanied by a progressive increase in the quantity of nuclear membrane associated condensed chromatin, an increase in the number of a granules, a progressive development of the DMS, and a reduction in the number of ribosomes, RER, and mitochondria. Megakaryocyte maturation also is accompanied by the formation of increasing quantities of glycogen in the cytoplasm; the glycogen particles often are found in large clumps. The DMS is an extensive system of membrane-lined cytoplasmic sacs, which arises as invaginations of the surface membrane; it demarcates areas of cytoplasm that eventually become platelets (Figure 33.51).
 |
Figure 33.49 Strong -naphthyl acetate esterase activity in a normal megakaryocyte. |
 |
Figure 33.50 Electron micrograph of a granular megakaryocyte from normal bone marrow. The cytoplasm contains a lymphocyte that appears to be traveling through the megakaryocyte (emperipolesis). |
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.
 |
Figure 33.51 Electron micrograph of a part of the intermediate zone of the cytoplasm of a granular megakaryocyte, showing the extensive demarcation membrane system, demarcating granule-containing future platelet areas. |
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.
 |
Figure 33.52 Electron micrograph of a platelet from normal blood. The platelet has been sectioned near, rather than at, the equatorial plane and, consequently, shows only part of the circumferential band of microtubules (arrow). The section also shows the electron-lucent vesicles of the surface-connected canalicular system, several platelet granules, a few mitochondria, and numerous clumps of glycogen molecules. |
P.828
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
P.829
show a positive paranuclear dot when stained for -naphthyl butyrate esterase; this staining is unaffected by fluoride. A substantial proportion of normal lymphocytes show either a paranuclear dot or diffuse granular positivity when stained for acid phosphatase. A paranuclear dot is found in both T cells and B cells but more frequently in T cells.
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.
 |
Figure 33.53 Various appearances of plasma cells in a smear of normal bone marrow. A prominent pale paranuclear zone and cytoplasmic vacuoles are seen in (A) and (B). The cytoplasm in (C) has a reticular appearance. The other cells in (A) are a nonphagocytic reticular cell and a late polychromatic erythroblast. |
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
P.830
and are often lined by RER. Many of these inclusions are rounded, elliptical, or irregular in outline, but a few are rhomboidal or needlelike and have a crystalline structure (Figure 33.55B D). Thus, the various types of cytoplasmic inclusion seen under the light microscope appear to be formed by the accumulation of unusually large quantities of immunoglobulin within regions of the RER.
 |
Figure 33.54 Electron micrograph of a plasma cell from a normal bone marrow showing numerous parallel sacs of rough endoplasmic reticulum and a very prominent Golgi apparatus immediately adjacent to the nucleus. The nucleus has moderate quantities of condensed chromatin. |
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.
P.831
 |
Figure 33.55 Different ultrastructural appearances of plasma cells from normal bone marrow. A. Cell with a prominent nucleolus, small quantities of condensed chromatin, several perinuclear mitochondria, some electron-dense material within all the sacs of rough endoplasmic reticulum (RER), and a single large, round, relatively electron-lucent intracytoplasmic inclusion lined by RER. B. Cell with multiple rounded or elliptical electron-dense intracytoplasmic inclusions lined by RER. C. Cell with two polygonal inclusions lined by RER. D. Cell with needlelike crystalline inclusions. The inclusions in (A C) probably result from the accumulation of large quantities of altered immunoglobulin within sacs of RER. |
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).
P.832
 |
Figure 33.56 Electron micrograph showing part of the Golgi apparatus and some of the sacs of RER from the plasma cell in Figure 33.54, at higher magnification. Four mitochondria are also present. |
 |
Figure 33.57 Neutrophil promyelocytes/myelocytes, metamyelocytes, stab cells, and granulocytes in a section of a paraffin-embedded trephine biopsy core from a hematologically normal subject. The two cells with large orange granules belong to the eosinophil granulocyte series. (H&E.) |
 |
Figure 33.58 Histochemistry of neutrophil series. A. Section of a paraffin-embedded marrow fragment from a hematologically normal subject showing cytoplasmic chloroacetate esterase activity in neutrophil promyelocytes/myelocytes and metamyelocytes but not in two erythroblasts (Leder's stain). B. Section of a paraffin-embedded trephine biopsy core showing PAS positivity in neutrophil granulocytes and their precursors. |
 |
Figure 33.59 Section of a paraffin-embedded trephine biopsy core from a hematologically normal adult showing a group of early and late polychromatic erythroblasts with halos around their nuclei. (H&E.) |
 |
Figure 33.60 Immunohistochemical demonstration of erythroblasts in a section of a paraffin-embedded trephine biopsy core. The section was reacted with antiglycophorin A antibody and the reaction visualized using an immunoalkaline phosphatase method. Courtesy of Dr. Alex Rice.) |
P.833
 |
Figure 33.61 Giemsa-stained section of a paraffin-embedded marrow fragment from a patient with erythroid hyperplasia due to a congenital dyserythropoietic state. The cell in the center with deep blue cytoplasm and prominent nucleoli is a proerythroblast. The photomicrograph also shows a few other proerythroblasts, several basophilic erythroblasts, and some early and late polychromatic erythroblasts. |
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.
 |
Figure 33.62 Two megakaryocytes from a section of a paraffin-embedded sample of clotted normal marrow (clot section). The cells in (A) showing a slight orange tinge are eosinophils and their precursors. The megakaryocyte in (B) displays emperipolesis. (H&E.) |
 |
Figure 33.63 Semithin section of an undecalcified, plastic-embedded trephine biopsy core showing a megakaryocyte and adjacent marrow cells. When compared with Figure 33.62A, this semithin section shows considerably more cellular detail, especially nuclear detail. The cytoplasmic granules of cells of the eosinophil series are clearly seen; these are stained red-orange. (H&E.) |
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).
P.834
Table 33.2 Differential Countsa on Marrow Smears from 28 Healthy Adults aged between 20 and 29 years | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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).
References
1. Preston SL, Alison MR, Forbes SJ, Direkze NC, Poulsom R, Wright NA. The new stem cell biology: something for everyone. Mol Pathol 2003;56:86 96.
P.835
2. Rywlin AM. Histopathology of the Bone Marrow. Boston: Little, Brown & Co.; 1976.
3. Krause JR, ed. Bone Marrow Biopsy. Edinburgh: Churchill Livingstone;1981.
4. Bartl R, Frisch B. Normal bone marrow: histology, histochemistry and immunohistochemistry. In: Wickramasinghe SN, McCullough J, eds. Blood and Bone Marrow Pathology. Edinburgh: Churchill Livingstone; 2003:53 69.
5. Andrade RE, Wick MR, Frizzera G, Gajl-Peczalska KJ. Immunophenotyping of hematopoietic malignancies in paraffin sections. Hum Pathol 1988;19:394 402.
6. Pulford KA, Erber WN, Crick JA, et al. Use of monoclonal antibody against human neutrophil elastase in normal and leukaemic myeloid cells. J Clin Pathol 1988;41:853 860.
7. Gatter KC, Cordell JL, Turley H, et al. The immunohistological detection of platelets, megakaryocytes and thrombi in routinely processed specimens. Histopathology 1988;13:257 267.
8. Kubic VL, Brunning RD. Immunohistochemical evaluation of neoplasms in bone marrow biopsies using monoclonal antibodies reactive in paraffin-embedded tissue. Mod Pathol 1989;2:618 629.
9. van der Valk P, Mullink H, Huijgens PC, Tadema TM, Vos W, Meijer CJ. Immunohistochemistry in bone marrow diagnosis. Value of a panel of monoclonal antibodies on routinely processed bone marrow biopsies. Am J Surg Pathol 1989;13:97 106.
10. Walls AF, Jones DB, Williams JH, Church MK, Holgate ST. Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase. J Pathol 1990;162:119 126.
11. Horny HP, Wehrmann M, Steinke B, Kaiserling E. Assessment of the value of immunohistochemistry in the subtyping of acute leukemia on routinely processed bone marrow biopsy specimens with particular reference to macrophage-associated antibodies. Hum Pathol 1994;25:810 814.
12. te Velde J, Burkhardt R, Kleiverda K, Leenheers-Binnendijk L, Sommerfeld W. Methyl-methacrylate as an embedding medium in histopathology. Histopathology 1977;1:319 330.
13. Burkhardt R. Bone marrow histology. In: Catovsky D, ed. The Leukemic Cell (Methods in Hematology). Edinburgh: Churchill Livingstone; 1981:49 86.
14. Frisch B, Lewis SM, Burkhardt R, Bartl R. Biopsy Pathology of Bone and Bone Marrow. London: Chapman & Hall; 1985.
15. Frisch B, Bartl R. Atlas of bone marrow pathology. In: Gresham GA, ed. Current Histopathology. Vol 15. Dordrecht, The Netherlands: Kluwer Academic; 1990.
16. Beckstead JH, Halverson PS, Ries CA, Bainton DF. Enzyme histochemistry and immunohistochemistry on biopsy specimens of pathologic human bone marrow. Blood 1981;57:1088 1098.
17. Beckstead JH. The bone marrow biopsy: a diagnostic strategy. Arch Pathol Lab Med 1986;110:175 179.
18. Archimbaud E, Islam A, Preisler HD. Immunoperoxidase detection of myeloid antigens in glycolmethacrylate-embedded human bone marrow. J Histochem Cytochem 1987;35:595 599.
19. Burkhardt R. Pr parative Voraussetzungen zur klinischen Histologie des menschlichen Knochenmarks. I. Mitteilung. Blut 1966;13:337 357.
20. Burkhardt R. Pr parative Voraussetzungen zur klinischen Histologie von Knochenmark und Knochen. II. Ein neues Verfahren zur histologischen Pr paration von Biopsien aus Knochenmark und Knochen. Blut 1966;14:30 46.
21. Burkhardt R. Bone Marrow and Bone Tissue. Color Atlas of Clinical Histopathology. Berlin: Springer-Verlag; 1971.
22. Wood GS, Warnke RA. The immunologic phenotyping of bone marrow biopsies and aspirates: frozen section techniques. Blood 1982;59:913 922.
23. Falini B, Martelli MF, Tarallo F, et al. Immunohistological analysis of human bone marrow trephine biopsies using monoclonal antibodies. Br J Haematol 1984;56:365 386.
24. Hayhoe FGJ, Quaglino D. Haematological Cytochemistry. 2nd ed. Edinburgh: Churchill Livingstone; 1988.
25. Wickramasinghe SN. Human Bone Marrow. Oxford: Blackwell Scientific; 1975.
26. Gordon MY. Human haemopoietic stem cell assays. Blood Rev 1993;7:190 197.
27. Ogawa M. Hematopoiesis. J Allergy Clin Immunol 1994;94:645 650.
28. Baron MH. Hemangioblasts in adults? Blood 2004;103:1
29. Verfaillie C. Regulation of hematopoiesis. In: Wickramasinghe SN, McCullough J, eds. Blood and Bone Marrow Pathology. Edinburgh: Churchill Livingstone; 2003:71 85.
30. Krause DS. Regulation of hematopoietic stem cell fate. Oncogene 2002;21:3262 3269.
31. Lord BI, Dexter TM, ed. Growth factors in haemopoiesis. Baillieres Clin Haematol 1992;5.
32. Brenner M, ed. Cytokines and growth factors. Baillieres Clin Haematol 1994;7.
33. Moore MA, Metcalf D. Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br J Haematol 1970;18:279 296.
34. Dzierzak E, Medvinsky A, de Bruijn M. Qualitative and quantitative aspects of haematopoietic cell development in the mammalian embryo. Immunol Today 1998;19:228 236.
35. Palis J, Robertson S, Kennedy M, Wall C, Keller G. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 1999;126:5073 5084.
36. Bloom W, Bartelmez GW. Hematopoiesis in young human embryos. Am J Anat 1940;67:21 53.
37. Kelemen E, Calvo W, Fliedner TM. Atlas of Human Hemopoietic Development. Berlin: Springer-Verlag; 1979.
38. Gilmour JR. Normal haemopoiesis in intrauterine and neonatal life. J Pathol Bacteriol 1941;52:25 55.
39. Emura I, Sekiya M, Ohnishi Y. Two types of immature erythrocytic series in the human fetal liver. Arch Histol Jpn 1983;46:631 643.
40. Kalpaktsoglou PK, Emery JL. Human bone marrow during the last three months of intrauterine life. A histological study. Acta Haematol 1965;34:228 238.
41. Piney A. The anatomy of the bone marrow with special reference to the distribution of the red marrow. Br Med J 1922;2:792 795.
42. Custer RP, Ahlfeldt FE. Studies on the structure and function of bone marrow. II. Variations in cellularity in various bones with advancing years of life and their relative response to stimuli. J Lab Clin Med 1932;17:960 962.
43. Wickramasinghe SN, McCullough J, eds. Blood and Bone Marrow Pathology. Edinburgh: Churchill Livingstone; 2003.
44. Munka V, Gregor A. Lymphatics and bone marrow. Folia Morphol (Praha) 1965;13:404 412.
45. Bain BJ, Clark DM, Lampert IA, Wilkins BS. Bone Marrow Pathology. 3rd ed. Oxford: Blackwell Science; 2001.
46. Branemark PI. Bone marrow microvascular structure and function. Adv Microbiol 1968;1:1 65.
47. De Bruyn PPH, Breen PC, Thomas TB. The microcirculation of the bone marrow. Anat Rec 1970;168:55 68.
48. Wickramasinghe SN. Observations on the ultrastructure of sinusoids and reticular cells in human bone marrow. Clin Lab Haematol 1991;13:263 278.
49. Miller MR, Kasahara M. Observations on the innervation of human long bones. Anat Rec 1963;145:13 17.
50. Dominici M, Pritchard C, Garlits JE, Hofmann TJ, Persons DA, Horwitz EM. Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation. Proc Natl Acad Sci U S A 2004;101:11761 11766.
51. Benayahu D, Horowitz M, Zipori D, Wientroub S. Hemopoietic functions of marrow-derived osteogenic cells. Calcif Tissue Int 1992;51:195 201.
52. Chambers TJ. Regulation of osteoclast development and function. In: Rifkin BR, Gay CV, eds. Biology and Physiology of the Osteoclast. Boca Raton, FL: CRC Press; 1992:105 128.
53. Sturgeon P. Volumetric and microscopic pattern of bone marrow in normal infants and children. III. Histologic pattern. Pediatrics 1951;7:774 781.
54. Hartsock RJ, Smith EB, Petty CS. Normal variations with aging of the amount of hematopoietic tissue in bone marrow from the anterior iliac crest. A study made from 177 cases of sudden death examined by necropsy. Am J Clin Pathol 1965;43:326 331.
55. Tavassoli M, Eastlund DT, Yam LT, Neiman RS, Finkel H. Gelatinous transformation of bone marrow in prolonged self-induced starvation. Scand J Haematol 1976;16:311 319.
P.836
56. Gale E, Torrance J, Bothwell T. The quantitative estimation of total iron stores in human bone marrow. J Clin Invest 1963;42:1076 1082.
57. Trubowitz S, Masek B. A histochemical study of the reticuloendothelial system of human marrow its possible transport role. Blood 1968;32:610 628.
58. Burgio VL, Magrini U, Ciardelli L, Pezzoni G. An enzyme-histochemical approach to the study of the human bone-marrow stroma. Acta Haematol 1984;71:73 80.
59. Westen H, Bainton DF. Association of alkaline-phosphatase-positive reticulum cells in bone marrow with granulocytic precursors. J Exp Med 1979;150:919 937.
60. Tanaka Y. An electron microscopic study of non-phagocytic reticulum cells in human bone marrow. I. Cells with intracytoplasmic fibrils. Nippon Ketsueki Gakkai Zasshi 1969;32:275 286.
61. Biermann A, Graf von Keyserlingk D. Ultrastructure of reticulum cells in the bone marrow. Acta Anat (Basel) 1978;100:34 43.
62. Tsai S, Patel V, Beaumont E, Lodish HF, Nathan DG, Sieff CA. Differential binding of erythroid and myeloid progenitors to fibroblasts and fibronectin. Blood 1987;69:1587 1594.
63. Broudy VC, Zuckerman KS, Jetmalani S, Fitchen JH, Bagby GC Jr. Monocytes stimulate fibroblastoid bone marrow stromal cells to produce multilineage hematopoietic growth factors. Blood 1986;68:530 534.
64. Kirshenbaum AS, Kessler SW, Goff JP, Metcalfe DD. Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells. J Immunol 1991;146:1410 1415.
65. Zucker-Franklin D, Grusky G, Hirayama N, Schnipper E. The presence of mast cell precursors in rat peripheral blood. Blood 1981;58:544 551.
66. Denburg JA, Richardson M, Telizyn S, Bienenstock J. Basophil/mast cell precursors in human peripheral blood. Blood 1983;61:775 780.
67. Kempuraj D, Saito H, Kaneko A, et al. Characterization of mast cell-committed progenitors present in human umbilical cord blood. Blood 1999;93:3338 3346.
68. Rywlin AM, Ortega RS, Dominguez CJ. Lymphoid nodules of bone marrow: normal and abnormal. Blood 1974;43:389 400.
69. Rheingold JJ, Wislocki GB. Histochemical methods applied to hematology. Blood 1948;3:641 655.
70. Gibb RP, Stowell RE. Glycogen in human blood cells. Blood 1949;4:569 579.
71. Rozenszajn L, Leibovich M, Shoham D, Epstein J. The esterase activity in megaloblasts, leukaemic and normal haemopoietic cells. Br J Haematol 1968;14:605 610.
72. Scott RE, Horn RG. Ultrastructural aspects of neutrophil granulocyte development in humans. Lab Invest 1970;23:202 215.
73. Bainton DF, Ullyot JL, Farquhar MG. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J Exp Med 1971;134:907 934.
74. Cawley JC, Hayhoe FGJ. Ultrastructure of Haemic Cells. A Cytological Atlas of Normal and Leukaemic Blood and Bone Marrow. London: WB Saunders; 1973.
75. Bessis M. Living Blood Cells and Their Ultrastructure. Berlin: Springer-Verlag; 1973.
76. Parwaresch MR. The Human Blood Basophil. Berlin: Springer-Verlag; 1976.
77. Scott RE, Horn RG. Fine structural features of eosinophile granulocyte development in human bone marrow. J Ultrastruct Res 1970;33:16 28.
78. Leder LD. The origin of blood monocytes and macrophages. A review. Blut 1967;16:86 98.
79. Wickramasinghe SN, Hughes M. Globin chain precipitation, deranged iron metabolism and dyserythropoiesis in some thalassaemia syndromes. Haematologia (Budap) 1984;17:35 55.
80. Wickramasinghe SN, Lee MJ, Furukawa T, Eguchi M, Reid CD. Composition of the intra-erythroblastic precipitates in thalassaemia and congenital dyserythropoietic anaemia (CDA): identification of a new type of CDA with intra-erythroblastic precipitates not reacting with monoclonal antibodies to - and -globin chains. Br J Haematol 1996;93:576 585.
81. N mec J, Pol k H. Erythropoietic polyploidy. I. The morphology of polyploid erythroid elements and their incidence in healthy subjects. Folia Haematol Int Mag Klin Morphol Blutforsch 1965; 84:24 40.
82. Queisser U, Queisser W, Spiertz B. Polyploidization of megakaryocytes in normal humans, in patients with idiopathic thrombocytopenia and with pernicious anaemia. Br J Haematol 1971;20:489 501.
83. Jean G, Lambertenghi-Deliliers G, Ranzi T, Poirier-Basseti M. The human bone marrow megakaryocyte. An ultrastructural study. Haematologia (Budap) 1971;5:253 264.
84. Breton-Gorius J, Reyes F. Ultrastructure of human bone marrow cell maturation. Int Rev Cytol 1976;46:251 321.
85. Breton-Gorius J. The value of cytochemical peroxidase reactions at the ultrastructural level in haematology. Histochem J 1980;12:127 137.
86. Breton-Gorius J, Gourdin MF, Reyes F. Ultrastructure of the leukemic cell. In: Catovsky D, ed. The Leukemic Cell (Methods in Hematology). Edinburgh: Churchill Livingstone; 1981:85 128.
87. Larsen TE. Emperipolesis of granular leukocytes within megakaryocytes in human hemopoietic bone marrow. Am J Clin Pathol 1970;53:485 489.
88. Rozman C, Vives-Corrons JL. On the alleged diagnostic significance of megakaryocytic phagocytosis (emperipolesis). Br J Haematol 1981;48:510.
89. White JG. Current concepts of platelet structure. Am J Clin Pathol 1979;71:363 378.
90. Berndt MC, Castaldi PA, Gordon S, Halley H, McPherson VJ. Morphological and biochemical confirmation of gray platelet syndrome in two siblings. Aust N Z J Med 1983;13:387 390.
91. Thiele J, Wagner S, Weuste R, et al. An immunomorphometric study of megakaryocyte precursor cells in bone marrow tissue from patients with chronic myeloid leukemia (CML). Eur J Haematol 1990;44;63 70.
92. Al-Adhadh AN, Cavill I. Assessment of cellularity in bone marrow fragments. J Clin Pathol 1983;36:176 179.
93. Kerndrup G, Pallensen G, Melsen F, Mosekilde L. Histomorphometrical determination of bone marrow cellularity in iliac crest biopsies. Scand J Haematol 1980;24:110 114.
94. Bartl R, Frisch B. Biopsy of Bone in Internal Medicine: An Atlas and Sourcebook (Current Histopathology). Vol 21. Dordrecht, The Netherlands: Kluwer Academic; 1993.
95. Gairdner D, Marks J, Roscoe JD. Blood formation in infancy. Part I. The normal bone marrow. Arch Dis Child 1952;27:128 133.
96. Glaser K, Limarzi LR, Poncher HG. Cellular composition of the bone marrow in normal infants and children. Pediatrics 1950; 6:789 824.
97. Diwany M. Sternal marrow puncture in children. Arch Dis Child 1940;15:159 170.
98. Rosse C, Kraemer MJ, Dillon TL, McFarland R, Smith NJ. Bone marrow cell populations of normal infants: the predominance of lymphocytes. J Lab Clin Med 1977;89:1225 1240.
99. Steiner ML, Pearson HA. Bone marrow plasmacyte values in childhood. J Pediatr 1966;68:562 568.
100. Jacobsen KM. Untersuchungen ber das knochenmarkspunktat bei normalen individuen verschiedener altersklassen. Acta Med Scand 1941;106:417 446.
101. Young RH, Osgood EE. Sternal marrow aspirated during life. Cytology in health and in disease. Arch Intern Med 1935;55:186 203.
