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Home , Metamyelocyte, Monoblast, Myeloblast, Myelocyte, Precursor cell, Promyelocyte

NORMAL ANATOMY AND 1 HISTOLOGY OF

FETAL HEMATOPOIESIS regulatory factors. This tightly regulated process In the developing embryo and fetus, hema- involves self-renewal of hematopoietic stem topoiesis is first detected in the yolk sac and cells and the production of lineage-committed sequentially in the dorsal aorta (aorta-gonad- progenitor cells; the latter undergo a cascade of mesonephros), liver, spleen, and finally the differentiation resulting in the diverse mature bone marrow. In the yolk sac, hematopoiesis hematolymphoid elements that are released occurs in the 2nd and 3rd embryonic weeks into the (15,28). and is characterized by the sequential forma- The hematopoietic specificity of bone mar- tion of waves of hematopoietic progenitor cells row–derived stem cells has been challenged, with limited reconstitution capacity (2,8). This and a “plasticity” of these stem cells has been site is restricted to the exclusive production of postulated (18). Although controversial, some nucleated erythroid cells within blood islands; evidence suggests that bone marrow–derived these primitive nucleated erythrocytes are the stem cells may be induced to give rise to various first circulating cells of the developing embryo nonhematolymphoid lineages such as muscle, (2,4,6,8). The earliest multilineage hematopoi- cartilage, and nerve (18). The recent detection of etic precursor cells are found in the embryonic a second category of , the mesenchymal dorsal aorta (aorta-gonad-mesonephros/para- stem cell, has prompted reconsideration of this aortic splanchnopleura) (1,2,4,8). Specialized hypothesis. Consequently, current investigators adhesion molecules produced by stromal cells propose that mesenchymal stem cells are the within both the yolk sac and dorsal aorta progenitors of nonhematopoietic lineages, not promote hematopoiesis and angiogenesis (5). the hematopoietic stem cells (13,21,22). These Indeed, a common in utero for mesenchymal stem cells are a normal constitu- hematopoietic and endothelial cells has been ent of the bone marrow microenvironment and proposed and termed the hemangioblast (3,8). give rise to mesenchymal bone marrow stromal Stem cell migration to the fetal liver results constituents including fibroblasts, adipose cells, in hepatic hematopoiesis by about 6 weeks of endothelial cells, osteoblasts/osteocytes, and gestation, followed shortly thereafter (at about chondroblasts/chondrocytes (33). 7 weeks of gestation) by seeding of the fetal Hematopoietic stem cells are not specifi- spleen (1,6,7). Hematopoiesis is detected in cally identifiable by the traditional morpho- fetal bone marrow by 14 weeks of gestation, logic review of bone marrow aspirate smears, and bone marrow is the primary site of hema- although a resemblance to small lymphoid cells topoiesis throughout the rest of gestation. The is suggested. Instead, stem cells are defined by vertebral column is the most active site of fetal functional properties or immunophenotypic bone marrow hematopoiesis followed by femur, profile. Hematopoietic stem cells are capable of pelvis, fibula/tibia, and humerus (7). both self-renewal and multilineage differentia- tion, a proposed unique property of asymetric HEMATOPOIESIS cell division which results in one new stem cell and one daughter cell capable of differentiation Stem Cells (12,16). Immunophenotypic features of hema- The production of all hematolymphoid lin- topoietic stem cells include expression of CD34, eages within the bone marrow is achieved by c-kit, and Thy1, while both CD38 and specific lin- the coordinated interaction of hematopoietic eage markers are typically absent (18,20,27,28,35). stem cells, the bone marrow microenviron- Expression of CD34, other adhesion molecules, ment, and various stimulatory and inhibitory and chemokine and receptors is linked

1 Non-Neoplastic Disorders of Bone Marrow

to the localization (homing) of hematopoietic matopoietic stem cell activity are influenced by stem cells in highly specific bone marrow mi- these bone marrow microenvironment niches croenvironment niches (33). (16,19,24,30,33,36). T cells within the bone The homing of hematopoietic stem cells and marrow microenvironment play a key role in progenitor cells is regulated by , che- hematopoietic regulation by stimulating vari- mokines, and adhesion molecules (14,24,30). ous target cells to produce either stimulatory or Stromal cells provide survival and regulatory inhibitory regulatory factors. Similarly, mono- signals by direct contact with stem/progenitor cytes/ within the microenviron- cells within a microenvironmental niche (24). ment produce many cytokines such as interleu- Hematopoietic stem cells localize to the endos- kin (IL)1, IL6, colony-stimulating teal region of bone marrow; key signaling inter- factor (G-CSF), and –colony-stimulat- actions between hematopoietic stem cells and ing factor (M-CSF), all of which interact directly subendosteal osteoblasts account for this unique with hematopoietic stem cells and progenitor localization (12a,16,23,26,30,33). Recent studies cells (19). These regulatory factors may be se- suggest a major role for osteoblasts in support- creted into the extracellular matrix or remain ing hematopoiesis and regulating the size of the cell surface associated (11,33). pool (12a,16,30,33). Regulatory Factors The selective paratrabecular localization of im- mature granulocytic cells can be appreciated Numerous cytokines play a critical role in by morphologic and immunohistochemical as- hematopoiesis (Table 1-1). The majority of these sessment. In contrast, both lineage-committed cytokines are produced within the bone marrow progenitor cells and terminally-differentiated microenvironment by T cells, macrophages, cells tend to localize to central regions of the osteoblasts, and various other mesenchymal medullary cavity (29). stromal cells (33,34). By binding to specific mem- The transition from the undifferentiated sta- brane receptors on target cells, these cytokines tus of hematopoietic stem cells to committed initiate a cascade of intracellular genetic events lineage-specific differentiation is characterized that promote proliferation and maturation (9). by the regulation of expression of lineage-spe- Steady state hematopoiesis is maintained by both cific genes (27). The expression of these lineage- stimulatory and inhibitory regulatory factor pro- specific genes allows the committed progenitor duction as listed in Table 1-1. Some cytokines act cells to respond to stimuli generated from bone on the earliest hematopoietic stem cells, while marrow microenvironment elements, ultimate- others control the proliferation and maturation ly resulting in the production of various mature of lineage-committed progenitor cells. progeny (27). Lineage commitment is associated Stem cell factor (SCF) is a prototypic early with the loss of the self-renewal capability. acting cytokine that is critical not only for sustaining hematopoiesis but also for stem cell Bone Marrow Microenvironment migration and adhesiveness (10). In addition to The functional unit of hematopoiesis, the its action on hematopoietic stem cells through bone marrow microenvironment, includes the interaction with its cognate receptor c-kit, SCF capillary-venous sinus, the surrounding extra- is also critical for proliferation, matura- cellular matrix, and stromal elements. This is tion, and survival, accounting for its alternate a highly organized, three-dimensional matrix name of mast cell growth factor. Other early composed of structural scaffolding produced acting cytokines include FLT3 (FMS-like tyrosine by bone marrow stromal cells (fibroblasts, kinase 3) ligand and IL1, IL3, IL6, and IL11. macrophages, endothelial cells, adipocytes, Granulocyte-monocyte–colony-stimulating and reticular cells). The extracellular matrix factor (GM-CSF) is also a multilineage cytokine includes regulatory factors as well as fibronectin, whose major action is on granulocytic and collagen, laminin, and various proteoglycans erythroid colony forming units in (25,29,33). Interactions with the bone marrow assays. Prototypic late-acting cytokines include microenvironment determine the fate of he- G-CSF, erythropoietin, and thrombopoietin, matopoietic stem cells, since all aspects of he- which stimulate lineage-committed progenitor

2 Normal Anatomy and Histology of Bone Marrow

Table 1-1 HEMATOPOIETIC REGULATORY GROWTH FACTORSa Regulatory Factor Comments Early Acting Cytokines; Multilineage, Pleiotropic or Synergistic Growth Factors Stem cell factor Ligand for c-kit receptor In receptor tyrosine kinase superfamily Many other names: kit ligand, steel factor, mast cell growth factor Influences stem cell migration, adhesiveness, proliferation Required to support hematopoiesis Synergistic with other regulatory factors Granulocyte/monocyte colony-stimulating factor Multilineage cytokine (GM-CSF) Promotes cell survival by suppressing apoptosis Interleukins (IL) 1, 3, 4, 6, 7, 11, 12 Multilineage (including B and T cells) cytokines Synergistic with other interleukins; IL-6 critical for plasma cell production IL3 Multi-CSF, multilineage cytokine Promotes cell survival by suppressing apoptosis FLK2/FLT3 (FMS-like tyrosine kinase 3) ligand Multilineage growth factor in receptor tyrosine kinase superfamily Lineage-Specific Cytokines Granulocyte colony-stimulating factor (G-CSF) Proliferation, maturation of neutrophilic lineage Monocyte colony-stimulating factor (M-CSF) Proliferation, maturation of monocytic/ lineage IL5 Promotes , production Erythropoietin Proliferation, maturation of erythroid lineage Thrombopoietin Proliferation, maturation of megakaryocytic lineage Inhibitory Factors Transforming growth factor-beta (TGF-b) Mediates other negative regulatory factors Interferon-alpha (IFN-a) Nonlineage-specific inhibitory protein Tumor necrosis factor-alpha (TNF-a) Nonlineage-specific inhibitory protein Macrophage inflammatory protein-1alpha (MIP1a) Antagonizes positive regulatory factors Lactoferrin, transferrin Myeloid inhibitory proteins; suppress GM-CSF production by and T cells aData from references: 9, 10, 17, 25, 31, 32, and 34. cells to produce , erythrocytes, and the expression of numerous genes, including , respectively. those encoding for growth factors, growth By integrating the various stimulatory cy- factor receptors, adhesion molecules, and vari- tokines with the stages of hematopoiesis, the ous enzymes (54,59,65). Early transcriptional production of various lineages can be illustrated regulation of involves SPI-1/PU.1, schematically (fig. 1-1). An artistic rendition of RUNX1, and core binding factor gene expres- hematopoiesis with the architectural localiza- sion (42,68). G-CSF is the dominant regulatory tion of specific lineages and stromal elements factor influencing committed is presented in figure 1-2. differentiation into neutrophils (43). Terminal granulocytic differentiation also is the result LINEAGE DEVELOPMENT of a highly coordinated transcription program which promotes complete differentiation into Granulocytic Cells neutrophils, with the subsequent acquisition The production of mature granulocytic ele- of responsiveness to activating infection-medi- ments from hematopoietic stem cells is an ex- ated stimuli (64). Homeostasis is maintained by quisitely regulated process that is controlled by inhibitors of granulocytic proliferation such as abundant transcription factors. These regulate lactoferrin and transferrin (Table 1-1) (62).

3 Non-Neoplastic Disorders of Bone Marrow

Figure 1-1 SCHEMATIC OF HEMATOPOIESIS Schematic of hematopoiesis with stimulatory cytokines showing progression from pluripotential hematopoietic stem cell to broad lymphoid and myeloid lineages. Further differentiation results in the production of normal lymphoid and hematopoietic lineages as well as and dendritic cells. (IL = interleukin; CSF = colony-stimulating factor; EPO = erythropoietin; TPO = thrombopoietin.)

The earliest recognizable myeloid cell is the monocytic cells. However, CD117 is also present . The myeloblast is characterized by on immature lymphoid and erythroid cells and a blastic nucleus with dispersed chromatin and mature mast cells, while CD34 is expressed on variably prominent nucleoli; the cytoplasm is the earliest progenitor cells of multiple lineages, generally moderate in amount and either agranu- including lymphoid precursors (44). Despite a lar or only minimally granular (Table 1-2; figs. characteristic immunophenotypic profile, my- 1-3–1-5). are infrequent in normal eloblasts are best enumerated by morphologic bone marrow and can be highlighted by the assessment. Both CD34 and HLA-DR expression presence of patchy cytoplasmic myeloperoxi- diminish in conjunction with the morphologic dase positivity (fig. 1-6). Granulocytic lineage features of maturation, which include the succes- elements express various myeloid-specific an- sive condensation of nuclear chromatin in con- tigens including CD13 and CD33; myeloblasts junction with the simultaneous acquisition of can be distinguished from more mature granu- first, primary, and later, secondary and gelatinous locytic elements by expression of CD34 and granules within the cytoplasm (fig. 1-8) (46). human leukocyte antigen (HLA)-DR (fig. 1-7). Although maturation is a biologic continu- Another immunophenotypic “marker” of um, by convention, maturing granulocytic cells immaturity is CD117 (c-kit receptor) expression, are designated as , neutrophilic which is evident on immature granulocytic and , neutrophilic , band

4 Normal Anatomy and Histology of Bone Marrow

Figure 1-3 Figure 1-2 STAGES OF NEUTROPHILIC MATURATION NORMAL BONE MARROW The stages of myeloid maturation, including myeloblast Artistic rendition of normal bone marrow with key (center), promyelocytes, myelocytes, and bands showing architectural features highlighted. (S = sinus; E = erythroid progression from immature to mature granulocytic cells, colonies; BT = bony trabeculae; My = immature myeloid, para- are seen in a bone marrow aspirate (Wright stain). trabecular, perivascular; V = vessel; Oc = osteocyte; F = fat cell; Ob = osteoblast; M = -shedding .)

Figure 1-4 Figure 1-5 MYELOBLAST WITH AGRANULAR CYTOPLASM MYELOBLAST AND EARLY A myeloblast with agranular cytoplasm and a large im- This composite shows two late myeloblasts, early mature nucleus in a bone marrow aspirate (Wright stain). neutrophilic promyelocytes with eccentric nuclei, and the early appearance of primary granules (bone marrow aspirate; Wright stain). neutrophils, and segmented neutrophils (Table 1-2; fig. 1-9) (38,50). Myeloblasts typically ac- situated in the maturation storage compart- count for less than 3 percent of the total cells ment; here, cells are available for rapid release in a differential cell count, while the successive into the circulation (fig. 1-10). These normal stages are progressively more abundant, with localization niches can be highlighted by immu- band and mature segmented neutrophils the nohistochemical staining for myeloperoxidase, most numerous. Immature granulocytic cells which tends to be more intense in the primary are localized to paratrabecular and perivascular granule-rich, immature granulocytic cells (figs. regions of the hematopoietic cavity, while more 1-11, 1-12). mobilization is stimulated mature granulocytic cells are more centrally by a variety of factors including CXC chemokines

5 Non-Neoplastic Disorders of Bone Marrow

Figure 1-6 Figure 1-7 MYELOPEROXIDASE CYTOCHEMICAL STAIN IMMUNOHISTOCHEMICAL STAIN FOR CD34 Granular myeloperoxidase positivity (MPO) in the Immunohistochemical stain for CD34 shows a rare cytoplasm of myeloblasts (arrow) and maturing myeloid positive cell in normal elderly adult bone marrow. cells (bone marrow aspirate).

Figure 1-8 Figure 1-9 STAGES OF NEUTROPHILIC MATURATION STAGES OF NEUTROPHILIC MATURATION Progressive nuclear maturation from the round eccentric The stages of neutrophilic maturation in a bone marrow nucleus of a promyelocyte (P) (lower left) through the mye- aspirate include early promyelocyte (P), neutrophilic locyte (My), (Mt), band (B), and segmented neu- (My), neutrophilic metamyelocyte (Mt), band trophil (PMN) (bone marrow aspirate smear; Wright stain). neutrophil (B), and segmented neutrophil (PMN). The progression from basophilic to eosinophilic cytoplasm and the acquisition of first, primary, and then, secondary which are systemic inflammatory mediators granules in conjunction with gradual and progressive (40). Chemokine-mediated neutrophil mobiliza- nuclear segmentation and condensation of the nuclear tion is caused by the modulation of adhesion chromatin are evident (Wright stain). molecule expression (40). In addition to myeloperoxidase, primary granules also contain lysozyme, defensins, Secondary granules are first evident at the elastase, and acid hydrolases, along with other early neutrophilic myelocyte stage of matura- constituents (39,46). These primary (azurophil- tion; these granules contain lactoferrin, leuko- ic) granules are acquired at the myeloblast/pro- cyte alkaline phosphatase, vitamin B12-binding myelocyte stage of maturation and play a key proteins, lysozyme, and other constituents (fig. role in defense from microbial invasion and in 1-14) (46). These fine granules impart a pink, (fig. 1-13). subtly granular appearance to the cytoplasm of

6 Normal Anatomy and Histology of Bone Marrow

Table 1-2 GRANULOPOIESISa Stage of Maturation Morphology Cytochemical/Immunophenotypic Properties Myeloblast High nuclear to cytoplasmic ratio Myeloperoxidase + or – Blastic, dispersed chromatin Most myeloblasts are CD34+, HLA-DR+, and coexpress Agranular, minimally granular cytoplasm myeloid+ lineage antigen such as CD33 Promyelocyte Eccentric nucleus with prominent para- Myeloperoxidase + nuclear hof (pale zone) Typically CD34 –, HLA-DR –, and myeloid antigen + (e.g., Sparse, concentrated azurophilic granules CD33, CD13) Neutrophilic Round nucleus with condensed chromatin Myeloperoxidase +, leukocyte alkaline phosphatase + myelocyte Moderate to abundant secondary (specific) Myeloid antigen + (CD34 –, HLA-DR –) granules which give the cytoplasm a finely granular pink appearance Neutrophilic Indented nucleus, condensed chromatin Myeloperoxidase +, leukocyte alkaline phosphatase + metamyelo- Cytoplasm packed with granules with Myeloid antigen + (CD34-, HLA-DR-) cyte predominance of secondary granules

Band neutro- Horseshoe-shaped mature nucleus lacking Myeloperoxidase +, leukocyte alkaline phosphatase + phil discrete indentations Myeloid antigen + (CD34 – HLA-DR –) Cytoplasm packed with granules with pre- dominance of secondary granules; gelatinous granules also present Neutrophil 3-5 discrete nuclear lobes Myeloperoxidase+, leukocyte alkaline phosphatase + Highly condensed chromatin Myeloid antigen + (CD34 –, HLA-DR –) Cytoplasm packed with granules with pre- dominance of secondary granules; gelatin- ous granules also present aData from references 39, 46, and 62.

Figure 1-10 LOCALIZATION OF NEUTROPHILIC CELLS This composite shows the para- trabecular localization of immature myeloid elements (left), while more mature granulocytic cells are concentrated in central intra- medullary areas (right) (hema- toxylin and eosin [H&E] stain).

neutrophilic myelocytes, and persist through- The least well-recognized and most poorly out the subsequent maturation to neutrophils understood type of neutrophil granule is the (figs. 1-15, 1-16). Upon release, these granule gelatinase (tertiary) granule, which lacks both constituents appear to play a key role in neu- myeloperoxidase and lactoferrin, but contains trophil adhesion (46). gelatinase, acetyltransferase, and lysozyme

7 Non-Neoplastic Disorders of Bone Marrow

Figure 1-11 Figure 1-12 IMMUNOHISTOCHEMISTRY FOR MYELOPEROXIDASE IMMUNOCYTOCHEMISTRY FOR MYELOPEROXIDASE MPO stain of a bone marrow core biopsy section Brightly MPO-positive immature myeloid cells demonstrate highlights the tendency for the bright positive immature a normal perivascular distribution in this bone marrow core myeloid elements to reside adjacent to bony trabeculae. biopsy section.

Figure 1-13 COMPOSITE OF PROMYELOCYTES Primary granule formation begins in this prominent para- nuclear hof. The eccentric nucleus typifies a neutrophilic promyelocyte (bone marrow aspirate; Wright stain).

(39,46,49a,62). Maturation stages from neutro- tissues (61). The blood component of neutrophils philic myelocyte, neutrophilic metamyelocyte, includes both circulating and marginated pools band neutrophil, to segmented neutrophil are of relatively equal size (figs. 1-19, 1-20). Margin- characterized by terminal cytoplasmic matura- ated neutrophils are attached to endothelial tion and gradual nuclear condensation and cells, an initial step in tissue migration. Neu- segmentation (figs. 1-17, 1-18). trophils, largely via release of granule contents, The granulopoietic cycle within the bone play a major role in host defense from microbial, marrow takes 10 to 14 days, but can be acceler- especially bacterial, infections, in conjunction ated by pharmacologic doses of recombinant with other cytotoxic and phagocytic functions. cytokines, either G-CSF or GM-CSF (62). Daily In addition to the “procytokines” listed above, production rates for neutrophils exceed 2 x neutrophil homeostasis is maintained by the 109/kg, and neutrophils circulate only briefly coordinated interaction of inhibitory factors in the peripheral blood before migrating into such as lactoferrin (62).

8 Normal Anatomy and Histology of Bone Marrow

Figure 1-14 Figure 1-15 NEUTROPHILIC MYELOCYTE NEUTROPHILIC MYELOCYTE Secondary granule formation begins in the Golgi region Mature neutrophilic myelocyte (My) has pinkish highlighted by the paranuclear hof in this early neutrophilic cytoplasm characterized by dispersed secondary granules myelocyte (bone marrow aspirate; Wright stain). (bone marrow aspirate; Wright stain).

Figure 1-16 NEUTROPHILIC MATURATION Left: Composite of a neutro- philic myelocyte (center), neutro- philic metamyelocyte (top), and band neutrophil (bottom) in a bone marrow aspirate smear shows the progression of maturation changes of the nucleus and cyto- plasm. Right: Electron micrograph of a myelocyte shows primary and secondary granules (bone marrow aspirate; Wright stain).

Monocytic and Dendritic Cells and typical, isolated individual cell distribution. Monocytes/macrophages and various types Both GM-CSF and M-CSF play major roles in the of dendritic cells share a common progenitor induction of monocyte/macrophage production cell with (65). Monocytes and re- within the bone marrow (49). lated dendritic cells play a pivotal role in host Dendritic cells are related to monocytes/ defense from microbial pathogens, wound heal- macrophages and play a key role in the innate ing, angiogenesis, hematopoiesis, and various (56,63). Evidence suggests inflammatory reactions (49). Although ubiq- that these unique antigen-presenting cells may uitous in all organs, macrophages/ be derived from either myeloid or lymphoid pro- and dendritic cells are generally inconspicuous genitor cells (55,57,67). Dendritic cells are respon- in normal tissues due to their sparse numbers sible for T-cell activation in the generation of a

9 Non-Neoplastic Disorders of Bone Marrow

Figure 1-17 Figure 1-18 LATE STAGES OF NEUTROPHILIC MATURATION BAND NEUTROPHIL The neutrophilic metamyelocyte (Mt), band neutrophil Band neutrophils are characterized by a horseshoe- (B), and segmented neutrophil (PMN) evident in the center shaped nucleus without evidence of early constriction or of this bone marrow aspirate smear highlight the nuclear segmentation. Two band neutrophils in the center of the and cytoplasmic features of maturation (Wright stain). slide can be compared to the adjacent segmented neutrophil (bone marrow aspirate; Wright stain).

Figure 1-19 Figure 1-20 BAND AND MATURE NEUTROPHILS MATURE SEGMENTED NEUTROPHIL Band and mature neutrophils circulate in the peripheral A segmented neutrophil with a Barr body in the blood (patient on granulocytic colony-stimulating factor peripheral blood of a normal female (Wright stain). [G-CSF]; Wright stain.) primary immune response (67). Dendritic cells gray cytoplasm with sparse fine granules (fig. are defined more by their functional activities 1-21). Although monocytic may than by specific morphologic features, although be composed of and promono- immunophenotypic subsets are well described cytes, neither monoblasts nor (55,56). Normal ranges for absolute dendritic are typically evident in normal bone marrow. cell counts in blood specimens from healthy Morphologically defined from studies of related adults are very low: 40/µL or less (47). leukemias, prototypic monoblasts have round Circulating monocytes are identified by their to oval nuclei with dispersed, blastic nuclear morphologic features, which include large size, chromatin (fig. 1-22). The cytoplasm is charac- reniform or folded nuclei, and abundant pale teristically abundant and pale blue, with either

10 Normal Anatomy and Histology of Bone Marrow

Figure 1-21 MONOCYTES This peripheral blood com- posite shows mature circulating monocytes with abundant, vacuo- lated, slate blue-gray cytoplasm and a reniform/folded nuclear configuration (Wright stain).

Figure 1-22 Figure 1-23 LEUKEMIC LEUKEMIC PROMONOCYTES A leukemic monoblast has voluminous, slate blue-gray, Leukemic promonocytes have abundant cytoplasm and finely granular cytoplasm and an immature round nucleus folded, immature nuclei (Wright stain). (Wright stain). agranular or subtle, finely granular cytoplasm. ration to macrophages/histiocytes occurs. Fixed Promonocytes are slightly more mature and tissue macrophages have round nuclei, variably demonstrate a folded nuclear configuration prominent nucleoli, and abundant cytoplasm with a typically prominent and fairly which frequently contains ingested cellular ele- dispersed nuclear chromatin (fig. 1-23). The ments (figs. 1-24, 1-25). Macrophages are readily cytoplasm of promonocytes is abundant and apparent on bone marrow aspirate smears and similar to that of monoblasts. are concentrated within particles. In normal bone Mature monocytes comprise less than 5 per- marrow, macrophages are generally less obvious, cent of the total cells in normal bone marrow, unless they contain ingested material from cell and are morphologically similar to their blood turnover, i.e., tingible body type (fig. 1-26). counterpart cells (fig. 1-21). Once monocytes mi- Diffuse cytoplasmic positivity for alpha- grate from the peripheral blood to tissue, matu- naphthyl butyrate and alpha-naphthyl acetate

11 Non-Neoplastic Disorders of Bone Marrow

Figure 1-24 Figure 1-25 BONE MARROW MACROPHAGES BONE MARROW MACROPHAGE Several bone marrow macrophages as well as fat cells are Bone marrow macrophage shows abundant iron and an evident in the center of this hypocellular bone marrow aspirate ingested orthochromic normoblast nucleus. smear following induction chemotherapy (Wright stain).

Figure 1-26 Figure 1-27 MACROPHAGES INGESTING NUCLEAR DEBRIS NONSPECIFIC ESTERASE CYTOCHEMICAL STAIN Hypercellular bone marrow core biopsy shows dispersed Cytochemical staining for nonspecific esterase (alpha- macrophages (histiocytes) containing ingested nuclear debris naphthol butyrate) shows diffuse positivity in the monocyte. (H&E stain). esterase (so-called nonspecific esterases) can from either myeloid or lymphoid progenitor be demonstrated by cytochemical stains in cells (55,57). Dendritic cells are infrequent in all monocyte stages (fig. 1-27). Immunohis- bone marrow, and immunohistochemical tech- tochemical markers most commonly used to niques (a profile consisting of CD68-, CD123-, highlight monocytes/macrophages include and CD43-positive, myeloperoxidase-negative lysozyme, CD68 (KP1 or PG-M1 epitopes), and cells) are generally required for cell identifica- CD163. These immunohistochemical markers tion (fig. 1-29) (56). have good sensitivity but variable specificity for The bone marrow is the primary site of mono- monocytic differentiation (fig. 1-28) (41,52,58). cyte/macrophage/ production; the The unique immunophenotype of the dendritic primary growth factor mediators of monocyte cells varies by the specific cell type, and differ- production include M-CSF, GM-CSF, and IL3. ences in phenotype are based on derivation Monocytic production within bone marrow is

12 Normal Anatomy and Histology of Bone Marrow

Figure 1-28 IMMUNOHISTOCHEMICAL STAIN FOR CD68 CD68 immunohistochemical stain highlights increased benign macrophages in a bone marrow core biopsy.

Figure 1-29 IMMUNOHISTOCHEMICAL STAIN FOR CD1A AND S-100 PROTEIN This composite shows rare CD1a-positive cells (left) and slightly more numerous S-100 protein–positive cells (right) in a normal bone marrow core biopsy from 1-year-old female.

estimated to take about 2 to 3 days. The cells are Although poorly understood, a hybrid eosino- directly released into the circulation and do not phil/basophil precursor cell has been postulated, constitute a bone marrow reserve compartment while mast cells appear to be derived as a sepa- (66). Monocytes circulate in the blood for 8 to rate lineage (45). IL3 and IL5 drive eosinophil 12 hours. Like neutrophils, monocytes within production; IL3 also plays a major role in baso- blood may be either circulating or marginated phil and mast cell production, in conjunction (66). In addition to bone marrow macrophages with other factors (45). For example, SCF (mast and dendritic cells, osteoclasts represent anoth- cell growth factor) is a critical factor in the in- er monocyte lineage–derived cell that is crucial duction of mast cell production (45,60). for bone resorption and remodeling (discussed The distinctive granules of each of these later in this chapter). three granulocytic cell types allows for easy identification of , , and Other Granulocytic Cells mast cells on bone marrow aspirate smears or Other types of granulocytic cells noted in in blood, although mast cells do not typically blood and bone marrow include eosinophils, circulate in normal subjects. On bone marrow basophils, and, in bone marrow only, mast cells. aspirate smears, both eosinophilic myelocytes

13 Non-Neoplastic Disorders of Bone Marrow

Figure 1-30 Figure 1-31 EOSINOPHILIC MYELOCYTE BASOPHILIC MYELOCYTE Bone marrow aspirate smear contains an eosinophilic A basophilic myelocyte in a bone marrow aspirate smear myelocyte (left), mature eosinophil (center), and segmented (Wright stain). neutrophil (right) (Wright stain).

Figure 1-32 Figure 1-33 EOSINOPHIL BASOPHIL A mature bilobed eosinophil in a peripheral blood smear A mature segmented basophil in a peripheral blood (Wright stain). smear (Wright stain). and basophilic myelocytes may be evident Both basophils and mast cells are effectors of (figs. 1-30, 1-31). Eosinophil granules are large the allergic response and immediate hypersensi- and refractile, and contain major basic protein, tivity reactions largely via mediator-stimulated , eosinophil cationic granule release; the granules of each of these protein, and other factors (51). Eosinophils cells have similar contents, notably histamine typically have bilobed nuclei (fig. 1-32). Eo- and heparin (37,45). Both cell types are pres- sinophil degranulation ameliorates immediate ent in low numbers in bone marrow. Mature hypersensitivity reactions, and the granules basophils have segmented nuclei, which may be are also effective antihelminthic agents (51). obscured by the prominent coarse, purple-black In addition, eosinophils produce SCF (c-kit li- cytoplasmic granules (fig. 1-33). In contrast, gand/mast cell growth factor), a property that mast cells exhibit round to oval nuclei, and may be linked to the close interaction between the cytoplasmic granules are smaller than ba- eosinophils and mast cells (48). sophil granules and are more purplish in color.

14 Normal Anatomy and Histology of Bone Marrow

Figure 1-34 Figure 1-35 ELONGATED MAST CELL MAST CELL A darkly staining, elongated, purple mast cell is present The mast cell (upper right) in a bone marrow aspirate in a bone marrow aspirate smear. Granules partially obscure smear has an oval configuration and a small round mature the nucleus (Wright stain). nucleus (Wright stain).

Figure 1-36 Figure 1-37 INCREASED MAST CELLS IMMUNOHISTOCHEMICAL STAIN FOR TRYPTASE A bone marrow particle has increased and individually The tryptase stain highlights increased dispersed mast dispersed dark purple-black mast cells (bone marrow cells in a bone marrow core biopsy. aspirate; Wright stain).

the dispersed and perivascular distribution of Similar to basophils, mast cell granules may these cells in normal bone marrow core biopsy overlay the nucleus, often obscuring it. Mast sections (fig. 1-37) (37,45,53). cells may be round, elongate, or even spindle Erythroid Cells shaped on bone marrow aspirate smears and core biopsy sections (figs. 1-34, 1-35). In normal Similar to other lineages, the production of bone marrow aspirate specimens, mast cells are erythroid elements is a highly regulated process concentrated within the stroma of bone marrow in which committed progenitor cells differenti- particles (fig. 1-36). They are more difficult to ate along the erythroid lineage pathway under appreciate on core biopsy sections and are best the control of cytokines, growth factors, and identified by immunohistochemical assessment signals from the bone marrow microenviron- with either tryptase or CD117, which highlights ment (72,76,77). These signals are transduced to

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