Normal Bone Marrow

Although hematopoietic stem cells circulate in small numbers, hematopoiesis, in steady-state conditions in adult life, is largely confined to the bone marrow. All lymphopoietic and hematopoietic cells are ultimately derived from pluripotent hematopoietic stem cells—slowly cycling cells with a capacity for self-renewal. Pluripotent stem cells give rise to common lymphoid stem cells and multipotent myeloid stem cells. The multipotent myeloid stem cells give rise to lineage-committed progenitors. None of the stem cells or progenitor cells is morphologically recognizable. Such cells can be identified in vitro by their capacity for self-renewal and their ability to differentiate to produce cells of specific lineages. Some of them can also be putatively identified by flow cytometry, immunocytochemistry, and immunohistochemistry, detecting the expression of antigens characteristic of stem cells such as CD34, with or without CD38. Stem cells in the marrow are located in stem cell “niches” adjacent to either bone or blood vessels, where they have a close relationship with stromal cells. Cells beyond the stage of a lineage-committed progenitor can be recognized from cytologic as well as functional and immunophenotypic characteristics. Some platelets are produced from megakaryocytes that have entered the circulation and lodged in the lungs. With this exception, all mature blood cells in healthy adults are produced in the bone marrow by a process involving repeated cell division and cellular maturation ( Fig. 10-1 ).

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Diagrammatic representation of one proposed scheme of the stem cell hierarchy showing the growth factors thought to operate at each stage.

Alternative models of hematopoiesis have been proposed, including one in which the common erythroid and megakaryocytic progenitor arises directly from the pluripotent lymphoid-myeloid stem cell (PSC; also known as the common lymphoid-myeloid progenitor ) rather than from the common myeloid progenitor (CMP; also known as multipotent myeloid stem cells ). B, B lymphocyte; Baso, basophil; BFU, burst-forming unit; CFU, colony-forming unit; CLP, common lymphoid progenitor; DC, dendritic cell; E, erythroid; Eos, eosinophil; EPO, erythropoietin; FLT3L, ligand of FLT3; G, granulocyte (neutrophil); G-CSF, granulocyte colony-stimulating factor; GM, granulocyte-macrophage; GM-CSF, granulocyte-macrophage colony-stimulating factor; GMP, granulocyte-monocyte progenitor; IL, interleukin; M, macrophage; Mast, mast cell; MB, myeloblast; M-CSF , monocyte colony-stimulating factor; MDC, myeloid dendritic cell; Meg, megakaryocyte; MEP, myeloid-erythroid progenitor; MKB, megakaryoblast; MoB, monoblast; NK, natural killer; ProE, proerythroblast; SCF, stem cell factor; T, T lymphocyte; TNK, T/NK cell progenitor; TPO, thrombopoietin.

Hematopoiesis occurs in a specific bone marrow microenvironment, in cavities surrounded and traversed by bony spicules. The intertrabecular spaces are occupied by stroma and hematopoietic cells, with the two elements having a dynamic interrelationship. The stroma is composed of stromal cells and a matrix of proteins such as laminin, thrombospondin, and fibronectin. Recognizable stromal elements include blood vessels, nerves, fat cells, other mesenchymal cells (e.g., reticular cells, macrophages, fibroblasts), and a delicate fiber network. The fiber network is detectable on a reticulin stain; if graded 0 to 4, most normal subjects have grade 0 to 1 reticulin, but some have grade 2. If fibrosis is graded 0 to 3, as in the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, normal subjects are graded as 0. Reticulin is deposited preferentially around arterioles and adjacent to bony spicules. In normal bone marrow, collagen is not detectable on a hematoxylin-eosin (H&E) stain or a trichrome stain. The earliest recognizable granulocyte precursors—myeloblasts and promyelocytes—are located against the periosteum and in a band around arterioles. Myelocytes, metamyelocytes, and neutrophils are found progressively farther from the endosteum. Recognizable cells of eosinophil lineage do not show the same distribution; eosinophil myelocytes and eosinophils are more randomly distributed. The distribution of basophils is not known. Maturing erythroid cells and megakaryocytes are found more centrally in the intertrabecular space. Erythroblasts are clustered, forming erythroid islands in which erythroid cells of varying degrees of maturity surround a central macrophage. Megakaryocytes are found preferentially in relation to sinusoids, and serial sections of bone marrow show that part of the megakaryocyte cytoplasm abuts a sinusoid. They may form small clusters, but these comprise no more than two, or occasionally three, cells. Other cellular components of the bone marrow include mast cells, lymphocytes, plasma cells, monocytes, and macrophages. Normal bone marrow architecture is shown diagrammatically in Figure 10-2 .

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Diagrammatic representation of the topography of normal bone marrow.

Osteoclasts, osteoblasts, myeloblasts, and promyelocytes are adjacent to the spicule of bone. Deeper in the intertrabecular space are maturing cells of neutrophil lineage, erythroid islands with a central macrophage, and interstitial lymphocytes. Eosinophils and their precursors are apparently randomly scattered, plasma cells are interstitial or form a sheath around capillaries, and megakaryocytes abut on a sinusoid at one extremity of the cell.

The regulation of hematopoiesis is highly complex. It involves the interaction of adhesion molecules on hematopoietic cells with their ligands on stromal cells and the action of hematopoietic growth factors such as stem cell factor, interleukin (IL)-3, IL-4, IL-5, IL-6, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, monocyte colony-stimulating factor, erythropoietin, and thrombopoietin. Growth factors may be secreted locally by bone marrow stromal cells (e.g., granulocyte-macrophage colony-stimulating factor), or they may be secreted at distant sites (e.g., erythropoietin). The ultimate effects of growth factors on hematopoiesis are mediated by transcription factors. Through their regulation of gene expression, these proteins coordinate the many proliferation and differentiation signals that reach the cell and are important for establishing the ultimate characteristics and phenotype of the mature blood cell. Although most diagrams of hematopoiesis suggest that cellular differentiation is unidirectional along one lineage, recent evidence suggests that it may be possible to reprogram cells of one lineage to differentiate into another lineage by altering the expression of various transcription factors. It is not clear whether this takes place only under experimental conditions, in certain pathologic situations, or perhaps even occasionally in normal hematopoiesis. The stages at which various growth factors are thought to operate are shown in Figure 10-1 .

The proportions of different hematopoietic cells normally present in the bone marrow are best determined by examining bone marrow from healthy volunteers, but it is also possible to examine marrow obtained from volunteer patients who are apparently hematologically normal. Patients with normal blood counts who require surgery for conditions that are unlikely to have any influence of bone marrow activity are suitable. Differential counts can be performed on wedge-spread films prepared directly from the bone marrow aspirate, on buffy coat preparations, or on films of crushed marrow particles. For wedge-spread films, the first 0.1 to 0.2 mL of the aspirate should be used so that there is minimal dilution by peripheral blood. The effects of dilution should be further minimized by counting the trails behind individual particles. For films of crushed particles, dilution is less of a problem; however, the films may be thicker, so identification of individual cells is more difficult. Whether wedge-spread films, buffy coat preparations, or films of crushed particles are used, a large number of cells must be counted because some of the cells of interest are present in a low proportion, and the count would otherwise be very imprecise. The International Council for Standardization in Haematology (ICSH) recommends that at least 500 cells be analyzed whenever the cell percentages will be used for diagnostic purposes ; following this guidance is particularly important when the percentage will be used to assign a diagnostic category (e.g., acute myeloid leukemia versus myelodysplastic syndrome). The advice of the WHO expert group is the same. Results of studies with these methods are summarized in Table 10-1 . In one study, the myeloid-to-erythroid ratio was found to be higher in women than in men, but this was not confirmed in two other studies.

Bone marrow trephine biopsy sections should be examined systematically, assessing the adequacy of the specimen, the bone structure, the cellularity, all myeloid lineages, lymphocytes and plasma cells, blood vessels and stroma, and any abnormal infiltrate.

Bone marrow cellularity in health is dependent on the age of the subject. The proportion of the marrow cavity occupied by hematopoietic and lymphoid cells rather than adipose cells varies from 100% at birth to between 30% and 65% after age 80 years. Between ages 30 and 70 years, cellularity is of the order of 40% to 70%. Figure 10-3 shows a bone marrow biopsy section with normal cellularity in comparison with hypocellular and hypercellular bone marrow specimens.

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Bone marrow biopsy of normal cellularity (A) compared with hypocellular (B) and hypercellular (C) biopsies.

Hematopoiesis

Erythropoiesis

The morphologic features of erythroid precursors in bone marrow films and sections are summarized in Table 10-2 and illustrated in Figures 10-4 to 10-8 . In normal bone marrow, cells of each successive stage of maturation are more numerous than those of the preceding stage. Erythroid islands may be noted in bone marrow aspirates ( Fig. 10-9 ) but are more readily appreciated in trephine biopsy sections, where they are located in the intertrabecular space away from the surface of the bone ( Fig. 10-10 ). In trephine biopsy sections, an artifactual halo around erythroid nuclei can aid in their identification. In normal subjects, a low proportion of erythroblasts may show binuclearity, cytoplasmic bridging, detached nuclear fragments, and irregular hemoglobinization (see later).

Table 10-2

Cytologic Features of Erythroid Precursors in Bone Marrow Aspirates and Trephine Biopsy Sections

Cell	Bone Marrow Aspirate	Bone Marrow Trephine Biopsy Section *
Proerythroblast	Large round cell, 12-20 µm in diameter, with finely stippled or reticular chromatin pattern and strongly basophilic (deep blue) cytoplasm; one or more nucleoli, which may be indistinct; there may be a perinuclear clear Golgi zone	Large round cell with round or slightly oval nucleus and one or more visible nucleoli, which are often linear or irregular and may abut on the nuclear membrane; cytoplasmic basophilia is marked and is most readily detected on Giemsa stain
Early erythroblast (basophilic erythroblast)	Similar to proerythroblast but smaller, and some chromatin clumping is now apparent; hemoglobin synthesis starts at this stage, but cytoplasm still appears deeply basophilic; perinuclear Golgi zone may be apparent	Somewhat smaller than a proerythroblast, but otherwise with similar features
Intermediate erythroblast (polychromatic erythroblast)	Intermediate-sized cell with less basophilic cytoplasm than early erythroblast and lower nuclear-to-cytoplasmic ratio; moderate chromatin condensation into coarse clumps; paranuclear, often partly perinuclear Golgi zone may be apparent; if Golgi zone is paranuclear, nucleus may be somewhat eccentric	Intermediate-sized cell with less cytoplasmic basophilia than early erythroblast; moderate chromatin clumping; sections of paraffin-embedded biopsy specimens may exhibit artifactual perinuclear halo owing to cytoplasmic shrinking
Late erythroblast (sometimes called orthochromatic erythroblast )	Small cell, not much larger than erythrocyte, with lower nuclear-to-cytoplasmic ratio and less cytoplasmic basophilia than intermediate erythroblast; chromatin clumping is marked, and cytoplasm is acquiring a pink tinge owing to increasing amounts of hemoglobin; however, when erythropoiesis is normoblastic, there is still some cytoplasmic basophilia, so this cell is not truly orthochromatic	Small cell with condensed chromatin, pink (eosinophilic) cytoplasm on hematoxylin-eosin stain, little basophilia on Giemsa stain, and prominent perinuclear halo; nucleus is rounder and more regular than that of a lymphocyte

* Erythroblasts of various stages of maturation are found clustered around a macrophage to form an erythroid island.

Assessment of erythropoiesis in aspirate films requires not only a Romanowsky stain (e.g., Wright-Giemsa or May-Grünwald-Giemsa stain) but also a Perls Prussian blue stain; the latter both assesses storage iron and determines the presence, number, and distribution of erythroblast siderotic granules. A Perls stain identifies hemosiderin but not ferritin. Normal late erythroblasts have a small number of scattered fine hemosiderin granules ( Fig. 10-11 ). Occasional intermediate erythroblasts may also contain siderotic granules. A Perls stain on trephine biopsy sections is informative if specimens have been plastic embedded; storage iron can be assessed, and abnormal sideroblasts can be detected. A Perls stain on sections from a paraffin-embedded, decalcified biopsy specimen is much less reliable because storage iron may have been removed in whole or in part by the process of decalcification and, regardless of whether storage iron is present, siderotic granules cannot be assessed.

Granulopoiesis

The morphologic features of granulocytic (specifically neutrophil) precursors in bone marrow films and sections are summarized in Table 10-3 and illustrated in Figures 10-12 to 10-15 . Maturating cells of eosinophil and basophil lineage can be recognized morphologically from the myelocyte stage onward in aspirate films. When there is reactive eosinophilia, it is often possible to recognize eosinophil promyelocytes, cells with a persistent nucleolus and a paranuclear Golgi zone that have dark purple proeosinophilic granules and some mature granules with eosinophilic characteristics. In trephine biopsy sections, eosinophils, neutrophils, and their precursors can be identified, but cells of basophil lineage cannot be recognized because their granules are lost during processing.

Table 10-3

Cytologic Features of Granulocyte (Neutrophil) Precursors in Bone Marrow Aspirates and Bone Marrow Trephine Biopsy Sections

Cell	Bone Marrow Aspirate	Bone Marrow Trephine Biopsy Section
Myeloblast	Large cell, 12-20 µm in diameter, with high nuclear-to-cytoplasmic ratio, moderate cytoplasmic basophilia, and diffuse chromatin pattern, often with one or more round or oval nucleoli; myeloblast is more irregular in shape than proerythroblast, and its cytoplasm is less basophilic; there may be small numbers of azurophilic (reddish-purple) granules	Large cell with high nuclear-to-cytoplasmic ratio, located near the surface of a bony spicule or an arteriole; nucleolus is rounder than that of a proerythroblast and does not touch the nuclear membrane; on Giemsa stain, cytoplasmic basophilia is less than that of a proerythroblast
Promyelocyte	Larger cell than a myeloblast, 15-25 µm in diameter, with more plentiful basophilic cytoplasm and more abundant reddish-purple azurophilic or primary granules; paranuclear Golgi zone; eccentric nucleus containing a nucleolus	Larger cell than a myeloblast, with a similar nucleus but more abundant granular cytoplasm, similarly located near a bony spicule or an arteriole
Myelocyte	Medium-sized to large cell, 10-20 µm in diameter; nucleus lacks a nucleolus and shows some chromatin condensation; cytoplasm is more acidophilic (pinker) than that of a promyelocyte and contains azurophilic granules (which now stain less strongly), and the cytoplasm is acquiring a pink-lilac tinge due to the presence of granules below the level of resolution of the light microscope; The Golgi zone is not conspicuous, but its presence may lead to slight nuclear indentation	Smaller cell than a promyelocyte, located farther away from the bone surface; the cytoplasm is granular, and the oval nucleus has no apparent nucleolus
Metamyelocyte	Medium-sized cell, 10-12 µm in diameter; resembles myelocyte, with granular acidophilic cytoplasm but indented or U- shaped nucleus	Medium-sized cell resembling a myelocyte and similarly situated, but with an indented or U- shaped nucleus
Band form and neutrophil	Medium-sized cells with granular pink cytoplasm and band-shaped or segmented nucleus, respectively; chromatin is coarsely clumped, particularly in the mature neutrophil	Medium-sized cells located some distance from a bony spicule or an arteriole, with granular cytoplasm and coarsely clumped chromatin in band-shaped or lobulated nuclei

Megakaryocytes and Thrombopoiesis

Three stages of megakaryocyte maturation can be recognized in normal bone marrow. All recognizable normal megakaryocytes are large polyploid cells. The smallest immature megakaryocytes measure 30 µm or more in diameter and have a high nuclear-to-cytoplasmic ratio and basophilic, often “blebbed” cytoplasm. Mature megakaryocytes are large cells, up to 160 µm in diameter, generally with a lobulated nucleus and pink or lilac granular cytoplasm ( Fig. 10-16 ); sometimes platelets are apparent, budding from the surface. A late megakaryocyte ( Fig. 10-17 ) is similar in size to an immature megakaryocyte because virtually all cytoplasm has been shed as platelets, leaving only a rather pyknotic nucleus with a thin rim of cytoplasm. Caution should be exercised in interpreting cytologic features of megakaryocytes because these large cells are very prone to crushing during the spreading of a bone marrow film; this may fragment a nucleus or cause some parts of the nucleus to be partly extruded from the cell. The cytoplasm of megakaryocytes may appear to contain intact cells of other lineages; these are actually within the surface-connected canalicular system. This phenomenon, known as emperipolesis, is physiologic but may be exaggerated in various pathologic states.

In histologic sections, mature megakaryocytes are easily recognized by their large size, plentiful cytoplasm, and lobulated nuclei ( Fig. 10-18 ). They can be highlighted by a Giemsa stain, which also demonstrates platelet demarcation zones in the cytoplasm, or by a periodic acid–Schiff (PAS) stain, which shows glycogen-rich pink cytoplasm. Late megakaryocytes are readily recognized as apparently bare megakaryocyte nuclei, which are larger than the nuclei of bone marrow cells of any other lineage and are more pyknotic than other nuclei of comparable size. Early megakaryocytes can be more difficult to recognize because they are not much larger than other bone marrow cells, and their features are not very distinctive. They are more readily appreciated by immunohistochemistry with a monoclonal antibody directed at platelet antigens such as CD61 for platelet glycoprotein IIIa or CD41 for platelet glycoprotein IIb.

Megakaryocytes are irregularly distributed in the bone marrow, and determining whether the number of megakaryocytes in a bone marrow aspirate is normal is difficult and necessarily subjective; it often relies on the quality of the film as well as the experience of the observer. In bone core biopsy sections from hematologically normal subjects, there are usually three to six megakaryocytes in each intertrabecular space; clusters of three or more megakaryocytes are not normally seen. In normal bone marrow, megakaryocytes may be seen to abut on sinusoids and are not found in a paratrabecular position.

Other Myeloid Cells

Monocytes, macrophages, mast cells, and osteoclasts are all of myeloid origin. Low but variable numbers are recognized in the bone marrow of healthy subjects.

Monocytes and macrophages are a minor population in normal bone marrow aspirates. Macrophages may be seen as isolated cells or in relation to erythroblasts in an erythroid island. Macrophages may contain cellular debris or hemosiderin ( Fig. 10-19 ).

Normal mast cells, although infrequent, are readily recognizable in bone marrow aspirate films because of their distinctive cytologic features. They generally have central, round, or oval nuclei, and their cytoplasm is packed with brightly staining purple granules ( Fig. 10-20 ); the majority are round or oval, but a minority may be fusiform. In H&E-stained sections, the scattered mast cells present in normal bone marrow are not readily recognizable. However, they are easily demonstrated on a Giemsa stain, which stains their granules purple; they are preferentially located adjacent to bone and around arterioles but are also scattered in small numbers throughout the marrow. Mast cells can also be demonstrated by immunohistochemical stains such as mast cell tryptase.

Osteoclasts are large, generally multinucleated cells with quite heavily granulated cytoplasm ( Fig. 10-21 ). Their multiple nuclei are oval and very uniform in appearance; each has a single lilac nucleolus. Only small numbers are seen in aspirates from healthy adults, but they are more numerous in aspirates from children. In histologic sections, osteoclasts are apparent as multinucleated cells adjacent to bone ( Fig. 10-22 ). Occasionally, apparently mononuclear osteoclasts can be recognized from their position and cytologic features.

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