Toxicologic Pathology, 34:548565, 2006

C by the Society of Toxicologic Pathology

Copyright

ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1080/01926230600939856

Normal Structure, Function, and Histology of the Bone Marrow

GREGORY S. TRAVLOS
Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, North Carolina 27709, USA
ABSTRACT
While a complete blood count provides information regarding possible treatment-related effects reected in the peripheral blood, morphological
evaluation of bone marrow cytology and parafn sections provides information about bone marrow tissue architecture that otherwise would be missed
by examination of peripheral blood alone. In decalcied, parafn-embedded, hematoxylin and eosin (H&E)-stained sections of bone marrow, the
more mature stages of the erythroid and myeloid cells, adipocytes, mast cells, and megakaryocytes can be identied, but lymphoid cells as well as
immature progenitor cells can not be reliably identied. The quality of the marrow sections is governed by numerous variables related to specimen
collection and processing and must be considered. In addition to discussing normal structure, function, and histology of bone marrow, methods for
preparation and evaluation of bone marrow are presented.
Keywords. Lymphopoiesis; hematopoiesis; xation; decalcication; M:E ratio.

(Schermer, 1967), 2% in dogs (Jain, 1986b) and 5% in humans (Picker and Siegelman, 1999). The bone marrow is the
major hematopoietic organ, and a primary lymphoid tissue,
responsible for the production of erythrocytes, granulocytes,
monocytes, lymphocytes and platelets. A brief discussion of
bone marrow structure and function will be presented here;
detailed descriptions can be found elsewhere (Jain, 1986b,
Weiss and Geduldig, 1991; Wickramasinghe, 1992; Picker
and Siegelman, 1999; Hoffman et al., 2000; Abboud and
Lichtman, 2001).
The inner surface of the bone cavities and the outer surface of the cancellous bone spicules within the cavities are
covered by an endosteal lining consisting of a single layer of
at bone-lining cells supported by a thin layer of reticular
connective tissue; osteoblasts and osteoclasts are also found
within the endosteal lining (Figure 2).
In long bones, one or more nutrient canals (containing a
nutrient artery and 1 or 2 nutrient veins) pass through the cortical bone entering the marrow cavity obliquely. In at bones,
the marrow is served by numerous blood vessels of various
sizes entering the marrow via large and small nutrient canals.
After entry, the artery splits into ascending and descending
branches that run parallel to the long axis in the central part
of the marrow cavity, coiling around the primary venous marrow channel, the central longitudinal vein (Figure 3). These
artery branches give rise to a multitude of small thin-walled
arterioles (Figure 4) and capillaries that extend outwardly toward the cortical bone. Near the bone, the arterioles open
up and anastomose with a plexus of venous sinuses. These
venous sinuses drain via collecting venules that lead back
centrally to the central longitudinal vein that then drains via
the nutrient veins. The marrow has an extensive blood supply
(Figure 5). Also, it appears that nutrient artery-derived capillaries extend into the Haversian canals, return to the marrow
cavity then open into the venous sinuses. Thus, there is a circular pattern to blood ow within the marrow cavity, from
the center of the marrow cavity toward the periphery of the
marrow cavity then back toward the center. In long and at
bones, the blood supplies of the bone and bone marrow are

INTRODUCTION
Blood and bone marrow is one of the largest organs in the
body and is an important potential target organ of chemical
exposure (Lund, 2000). For example, it was suggested that
drug-related blood dyscrasias represented 10% of all blood
dyscrasias reported in Sweden, and, 40% of those resulted
in fatality (Bottinger and Westerholm, 1973). Since effects
of a compound may be elicited in the circulating blood cell
mass or the production of blood cells, evaluations of single
or serial whole blood samples and smears, bone marrow aspirates, and marrow tissue sections are needed to understand
the alterations in the leukon, erythron or thrombon that may
occur in toxicity studies. Examples of blood and bone marrow
toxicity can be found in Table 1.
Assessments of the blood and bone marrow have become
routine procedures in the investigation of hematologic disorders in toxicology and safety assessment studies. Evaluation
of blood has been extensively described (Jain, 1986a; Perkins,
1999; Ryan, 2001). The focus of this article will be evaluation of the bone marrow with the objectives of reviewing of
some concepts regarding the bone marrow structure and function and review of qualitative and quantitative bone marrow
evaluation methods. A review of various lesions of the bone
marrow in laboratory rats, mice and dogs will be presented
in a subsequent discussion (Travlos, 2006).
BONE MARROW STRUCTURE AND FUNCTION
The bone marrow is found within the central cavities of axial and long bones (Figure 1). It consists of hematopoietic tissue islands and adipose cells surrounded by vascular sinuses
interspersed within a meshwork of trabecular bone. It accounts for approximately 3% of the body weight in adult rats

Address correspondence to: Gregory S. Travlos, Laboratory of Experimental Pathology, NIEHS/NIH, 111 Alexander Dr., MD B3-06, Research
Triangle Park, NC 27709, USA; e-mail: travlos@niehs.nih.gov
This research was supported by the Intramural Research Program of the
NIH, National Institute of Environmental Health Sciences.

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TABLE 1.Examples of chemical agents causing toxicological effects in the bone marrow and/or blood.
Agent

Aspirin
Azidothymidine
2-Chloronitrobenzene
Cobalt
Cupric Sulfate
Estrogen
Glucocorticoids
Glucocorticoids
Heparin
-Irradiation
-Lactam antibiotics
-Methyldopa
-Methyldopa
Nitrites
Penicillin
Ristocetin
Tumor necrosis factor
Warfarin

Effect

Site of action

Decreased platelet aggregation

Megaloblastic anemia
Heinz body anemia
Erythrocytosis
Hypoproliferative anemia
Aplastic anemia
Neutrophila
Lymphopenia
Thrombocytopenia
Nonresponsive anemia
Neutropenia
Immune-mediated anemia
Thrombocytopenia
Heinz body anemia
Immune-mediated anemia
Thrombocytopenia
Hypoproliferative anemia
Blood loss anemia

Mechanism

Platelets
Bone marrow
Erythrocytes
Bone marrow
Bone marrow
Bone marrow
Systemic
Systemic
Platelets
Bone marrow
Neutrophils
Erythrocytes
Platelets
Erythrocytes
Erythrocytes
Platelets
Bone marrow
Liver

interconnected through an endosteal network of vessels. The

venous sinuses are thin-walled, consisting of a layer of at endothelial cells with little to no basement membrane. The marrow does not have lymphatic drainage (Munka and Gregor,
1965).
Bone marrow innervation occurs with myelinated and nonmyelinated nerves that enter through the nutrient canals.
Some innervation also occurs through epiphyseal and metaphyseal foramina. Nerve bundles follow the arterioles with
branches serving the smooth muscle of the vessles or, occasionally, terminating in the hematopoietic tissue amongst
hematopoietic cells.
The hematopoietic tissue consists of a variety of cell
types including, the blood cells and their precursors, adventitial/barrier cells, adipocytes, and macrophages. The
hematopoietic tissue cells are not randomly arranged but
demonstrate a particular organization within the tissue (Weiss
and Geduldig, 1991) (Figure 2). For hematopoiesis to occur
it must be supported by a microenvironment that is able to
recognize and retain hematopoietic stem cells and provide
the factors (e.g., cytokines) required to support proliferation,
differentiation and maturation of stem cells along committed lineages. The hematopoietic microenvironment consists
of adventitial reticular cells (e.g., barrier cells), endothelial
cells, macrophages, adipocytes, possibly, bone lining cells
(e.g., osteoblasts) and elements of the extracellular matrix. A
more detailed discussion of the organization and function of
the hematopoietic microenvironment can be found elsewhere
(Weiss and Geduldig, 1991; Hoffman et al., 2000; Gasper,
2000a, 2000b, 2000c; Abboud and Lichtman, 2001).
Hematopoiesis is a compartmentalized process within the
hematopoietic tissue with erythropoiesis taking place in distinct anatomical units (erythroblastic islands); granulopoiesis
occurs in less distinct foci and megakaryopoiesis occurs
adjacent to the sinus endothelium. Upon maturation, the
hematopoietic cells, regulated by the barrier cells, traverse the
wall of the venous sinuses to enter the bloodstream; platelets
are released directly into the blood from cytoplasmic processes of megakaryocytes penetrating through the sinus wall
into the sinus lumen. Details of the hematopoietic process
can be found elsewhere (Jain, 1986b; Hoffman et al., 2000;
Gasper, 2000a, 2000b, 2000c; Abboud and Lichtman, 2001).

Altered thromboxane metabolism

Altered DNA synthesis
Methemoglobin and Heinz body formation
Increased erythropoietin production
Iron deciency-like; altered iron metabolism
Decreased stem cells and response to erythropoietin
Decreased emigration/margination, increased release
Bedistribution, lympholysis
Immune-mediated
Stromal reaction-myelobrosis
Immune-mediated
Anti-erythrocyte autoantibodies
Anti-platelet autoantibodies
Methemoglobin and Heinz body formation
Anti-penicillin antibodies bind to coated erythrocytes
Intravascular agglutination
Decreased availability of iron
Decreased vitamin K-dependent clotting factor production

The production, differentiation, and maturation of blood

cells are regulated by humoral factors (Table 2). Some factors
(e.g., BPA/IL-3) act on the more primitive cells and have a
general action, while others (e.g., erythropoietin) act on later
progenitors of a specic cell line. The sources of hematopoietic factors vary. Erythropoietin is produced primarily in the
kidney with minor amounts from the liver and stimulates proliferation of committed erythrocytic progenitors and release
of immature red cells; high levels increase the rate of differentiation into erythrocyte progenitors. Burst promoting activity

TABLE 2.Examples of factors that stimulate hematopoiesis.

Stimulation of pluripotent cells
Stem cell factor
IL-6
Erythropoiesis
Burst promoting activity (BPA)
Erythropoietin
Granulocyte-macrophage colony stimulating factor (GM-CSF)
Thyroid hormone
Growth Hormone
Testosterone
Granulocytopoiesis
GM-CSF
Granulocytopoietin (G-CSF)
IL-1
IL-3
IL-5
Eosinophilopoietin
Basophilopoietin
Interferon (INF)
Tumor necrosis factor (TNF)
Monocytopoiesis
GM-CSF
Macrophage colony stimulating factor (M-CSF)
Monocytopoietin
IL-3
Lymphopoiesis
Thymic hormone
Lymphocyte mitogenic factor
B-cell growth factor
B-cell differentiation factor
IL-1
IL-2
IL-3
IL-4
Megakaryocytopoiesis
M-CSF
Thrombopoietin

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FIGURE 1.Representative examples of bone marrow cellularity in long and

axial bones of normal adult B6C3F1 mice. The marrow spaces contain islands
and clusters of hematopoietic cells admixed with adipocytes. (A) Distal femur.
(B) Sternum. (C) Vertebra.

(BPA) is produced by T-lymphocytes and macrophages. IL3 is produced by T-lymphocytes and myeloid cells and
may be the same macromolecule as BPA. Colony simulating factors are produced by a variety of cells, including macrophages/monocytes, broblasts, endothelial cells,

TOXICOLOGIC PATHOLOGY

lymphocytes, and placenta. Most interleukins, B-cell growth

factor, and B-cell differentiation factor are derived from Tlymphocytes. IL-1 is produced by macrophages. Hormones
also play a physiological role (Jain, 1986c). For example,
circulating erythrocyte counts increase or decrease following
gonad removal in female and male rats, respectively; administration of the respective sex hormones abrogated the effects
of gonadectomy. Additionally, bone marrow morphology was
altered following ovariectomy in female rats (Benayahu et al.,
2000). Hormones of the pituitary, adrenals, thyroid and gonads appear to participate in erythropoiesis by altering erythropoietin production and erythroid progenitor response to
other factors (Jain, 1986c). For example, androgens, thyroxine and growth hormone increase erythropoietin production;
estrogen has an inhibitory erythropoietic effect.
Hematopoietic tissue is also sensitive to external inuences
and can become suppressed in response to dietary restriction, malnutrition, chronic inammation, toxicity, and proliferative or neoplastic disorders (Jain, 1986d; Meierhenry,
1990; Wierda, 1990; Reagan, 1993; NTP, 1999; NIEHS,
1999, 2001; Lund, 2000; Weiss, 2000). In the rat, nutritional
status is an important factor (Meierhenry, 1990). For example, diet restriction sufcient to halt weight gain in young rats
decreased marrow erythroid elements by 50%, myeloid elements by 40%, and megakaryocytes by 20% (Brown, 1954).
Complete restriction for 7 days reduced marrow cellularity
by 30% (Furman and Gordon, 1955). Levin et al. (1993),
demonstrated that a severe (25% of control) diet restriction
for 2 weeks resulted in a relative erythrocytosis, lymphopenia, thrombocytopenia and bone marrow necrosis. And, it has
been reported that, in the rat, protein intake, rather than total
calories, is more important for maintenance of erythropoiesis
(Bethard et al., 1958).
Hematopoiesis is a continuous process, but can be separated into distinct stages (Figure 6). The rst stage involves
uncommitted (pluripotent) stem cells contained in the bone
marrow. These pluripotent cells have two primary functions.
First, they maintain their numbers by a process of selfrenewal and, secondly, they have the ability to give rise to
all hematopoietic cells (erythrocytes, granulocytes, lymphocytes, monocytes, and platelets). They also appear to be found
in greater numbers peripherally from the central axis, near
the bone lining cells (Weiss and Geduldig, 1991; Picker and
Siegelman, 1999; Gasper, 2000c).
Most of the understanding of hematopoietic proliferation
and maturation has been derived using an irradiated syngeneic mouse model. Irradiated mice infused with donor cells
give rise to hematopoietic foci in the spleen. In vivo, it was
demonstrated that the stem cell pool could be measured in
the rat and mouse (Till and McCulloch, 1961). Donor mouse
cells injected into an irradiated mouse formed nodules in
the spleen that could be visually counted. It was demonstrated that these splenic colonies were clones (Becker et al.,
1963) and that the cells within these colonies were capable
of self-renewal and differentiation into the major cell lines
(Till et al., 1964). These splenic colonies have been shown to
arise from a single pluripotent cell, which has been termed the
colony forming unit-spleen (CFU-S). Depending on need, the
bone marrow microenvironment and growth factors inuence
pluripotent stem cells to differentiate into committed stem
cells of either the myeloid or lymphoid series (multipotential

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FIGURE 2.Schema of IL-1-activated distal medial metaphyseal femoral hematopoietic murine marrow. Trabecular bone (leader terminates on a canaliculus
containing extensions of an osteocyte) is enclosed by a complex layer of diverse cells. Osteoblasts (osteobl) and an osteoclast (osteocl), at, simple, or two-layered
nonactivated bone-lining cells (blc) are present. Reticular cells branch from the surface of bone to the adventitial surface of vascular sinuses (sinus 2). Barrier
cells cover two sites on the surface of bone, and extend en bloc into the marrow. The barrier cells are activated, displaying organelles associated with intense
protein synthesis and secretion. From the dependent aspect of bone, massed barrier cells sweep in a crescent, deep into the marrow. The crescent of barrier cells
holds many hematopoietic cells, notably putative stem cells and differentiating megakaryoctes. On the crescents edge, barrier cells branch into the surrounding
hematopoietic cells meeting with rather loosely disposed, richly branched barrier cells lying among and supporting hematopoietic cells. Barrier cells, especially in
hematopoietic zones containing very early differentiating stages, may insinuate long, slender processes between and around endothelium and adventitial tunics. The
blood-marrow barrier is thereby augmented, impeding emigration and immigration of circulating cells, preventing premature release of immature hematopoietic cells
to the circulation. In contrast, proles of vascular sinuses (sinus 3) can be made entirely of a simple layer of barrier cells stretched quite thin, except at the perikaryon.
These lie in hematopoietic zones containing late differentiating forms ready for delivery to the circulation, their wall beset with blood cell-lled apertures. They are
structurally suited to facilitate delivery of blood cells to the circulation. From: Weiss, L. and Geduldig, U. Barrier cells: Stromal regulation of hematopoiesis and
blood cell release in normal and stressed murine bone marrow. Blood 1991; 78:975-990. Copyright American Society of Hematology, used with permission.

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FIGURE 3.The cross-section (A) and longitudinal section (B) from the femur of a male B6C3F1 control from a 28-day toxicity study shows a cellular rich bone
marrow. The arrow indicates the central vein, and the arrowheads identify representative venous sinuses. The asterisk in 3A identies an area of shrinkage artifact
associated with histologic processing of the bone. CB = cortical bone.

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FIGURE 4.This high magnication of the femur cross-section shown in 3A more clearly shows the central vein (CV), nutrient arteries (arrowheads), and venous
sinuses (arrows) within the cellular rich bone marrow.

stem cells), or the second stage of hematopoiesis. They

have a limited capacity for self-renewal, but have the potential to differentiate and develop mature progeny. Myeloid
stem cells are the multipotential colony forming unit for
granulocytes, erythrocytes, monocytes, and megakaryocytes
(CFU-GEMM). The third stage is when committed stem
cells, inuenced by various growth factors, differentiate into
lineage-specic progenitor cells. Progenitor cells exist in the
bone marrow for megakaryocytes (CFU-Meg), lymphocytes,
erythrocytes (BFU-E), eosinophils (CFU-Eos), and basophils
(CFU-Baso). It appears neutrophils and monocytes arise from
a common precursor (CFU-GM).
Lymphopoiesis occurs within the bone marrow microenvironment of adult mammals (Allen and Dexter, 1984). And
B-lineage cells derived from the marrow can be identied
by sequential changes in cell size and expression of immunoglobulin chains. Large pre-B cells are early precursors
and contain heavy chains within the cytoplasm (Landreth
and Kincade, 1984). Large pre-B cells (1013 microns)
divide at least once to produce smaller (<9 microns) pre-

B cells. With gene rearrangement, the small pre-B lymphocytes mature into B cells; they express K and A light chains
in the cytoplasm (Wierda, 1990). The sequence of proliferation/maturation of B-lymphopoiesis is regulated by soluble factors secreted by stromal cells (Picker and Siegelman, 1999) and is sensitive to disruption by myelotoxic
chemicals. For example, polyhydroxy metabolites of benzene (e.g., hydroquinone) have been shown to affect Blymphopoiesis in the marrow causing maturation arrest of
the B-cells at the pre-B cell stage (Wierda and Irons, 1982;
King et al., 1988). T-cell lymphopoiesis occurs in the thymus
that has been seeded with bone marrow-derived stem cells
(Le Douarin et al., 1984). There is some evidence indicating that the prothymocytes have undergone some differentiation and/or commitment prior to relocating to the thymus
(Picker and Siegelman, 1999). Morphologically, the rat or
mouse bone marrow did not have, nor develop, lymphoidcell aggregates or structures resembling follicles, even after immunization (Geldof et al., 1983). Further, while the
marrow appeared to have a suitable microenvironment for

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FIGURE 5.Diagrammatical representation of the vascular supply of the bone marrow. Adapted from: Abboud, C. N. and Lichtman, M. A. (2001) Structure of the
marrow and the hematopoietic microenvironment. In Williams Hematology, 6th edition. Copyright McGraw-Hill, used with permission. Adaptive drawing by David
Sabio. 6.Representation of the maturation progression of the multiple cellular lineages present in the bone marrow. CFU = colony forming unit; E = erythyroid;
Meg = megakaryocyte; Gemm = granulocytic, erythyroid, monocyte-macrophage, and megakaryocytic; GM = granulocyte/monocyte; G = granulocyte; M =
monocyte; Eo = eosinophil; Baso = basophil; L = lymphocyte. Drawing by David Sabio.

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immigrating antibody-producing cells, the cells dispersed

singly, in a random arrangement, and did not appear to contribute to the immune response.
METHODS FOR EVALUATING THE BONE MARROW
Evaluation of the hematopoietic system should be performed using a multi-pronged approach (i.e., peripheral blood
exam including a CBC and differential, bone marrow smear
exam or total femur counts and evaluation of cytocentrifuge
preparations and/or bone marrow histopathology). Bone marrow histopathology and examination of peripheral blood are
performed routinely in toxicity and safety assessment studies.
Cytological preparations can be made routinely but evaluations are generally reserved for instances in which hematological changes are identied and determination of the underlying cause is needed.
Histopathology is a subjective assessment and is useful for
evaluating bone marrow architecture, assessment of cellularity, estimation of M:E ratio (limited sensitivity), assessment
of cell lineages, estimation of iron stores and other features
(e.g., neoplasia, inammation, pigment, infectious agents).
Marrow smear evaluations and/or total femur counts provide quantitative results and better cellular morphology and
determination of M:E ratios and maturation indices. While
the following is a brief overview, more detailed information and techniques regarding histological and cytological
evaluation of bone marrow have been described (Lewis and
Rebar, 1979; Cline and Maronpot, 1985; Grindem, 1989;
Tyler and Cowell, 1989; Wickramasinghe, 1992; Buckley,
1995; Andrews, 1998; Car and Blue, 2000; Freeman, 2000;
Lanning, 2001; Valli et al., 2002).
When performing core marrow biopsies in adult dogs, it is
usually necessary to take samples from the iliac crest, sternum, proximal humerus, trochanteric fossa of the femur or
a rib, as the central femoral marrow cavity may be almost
entirely replaced by fat. In the rat and mouse, however, the
higher turnover of erythrocytes, due to a shorter circulating
life span, means that the marrow space in most bones remains populated for life. And, in the rodent, it appears that
the sternum and rib and, probably, humerus and proximal femur are important sampling sites, as the marrow at these sites
remains hematopoietically active regardless of the animals
age. For example, in the Fischer rat, from 4 months to 2 years
of age, the sternum, ribs, humerus and proximal femur had a
relatively similar marrow cellularity of approximately 68%
(Cline and Maronpot, 1985). The distal femur and proximal
tibia were similar at approximately 61% marrow cellularity.
Regardless of age, the distal tibia had a noticeable lack of
active hematopoiesis.
It is generally considered that, in the dog, normal bone marrow contains about 50% fat and 50% hematopoietic tissue;
approximately 70 to 80% of the marrow being hematopoietic
tissue in rats and mice (Valli et al., 2002). In the dog, however, marrow cellularity may range from 20% to 80% of the
marrow space, depending on site and age (Weiss, 1986; Valli
et al., 2002) (Figure 7). In a study evaluating Fischer rats,
depending on the age and anatomic site, the average marrow
space occupied by hematopoietic cells varied from 3388%
(Cline and Maronpot, 1985). In that study, the youngest animals had the highest marrow cellularity. For example, regardless of site, the mean marrow cellularity was 80% at

555

2 months of age; by 2 years of age, the cellularity decreased

to a mean of 66%. Examples of normal rat and mouse bone
marrow are shown in Figures 8 and 9.
In general, decalcied, parafn-embedded, hematoxylin
and eosin (H&E)-stained sections of bone marrow, the
more mature stages of the erythroid and myeloid cells,
adipocytes, mast cells, and megakaryocytes can be identied, but stem cells, immature myeloid, erythroid, lymphoid,
monocytoid and stromal cells cannot be identied consistently. An estimation of general hematopoietic activity and
the myeloid/erythroid ratio can also be performed. The erythroid elements are smaller with round, dense, and deeply
basophilic nuclei (Figure 10). The cytoplasm is basophilic in
the blast forms with increasing eosinophilia as they mature.
The granulocytes have large bean-shaped nuclei that are less
basophilic and more vesicular than the erythropoietic cells
(Figure 10). Megakaryocytes are easily recognized by their
large size and multilobulated nuclei (Figure 10). While mature lymphocytes can be identied in bone marrow smears
(Figure 12), unequivocal identication of lymphoid lineage
cells in decalcied H&E-stained sections of bone with bone
marrow is not readily accomplished.
For smears, marrow can be obtained from an exposed surface of, or extruded from, the sternum or rib using a sable
brush (e.g., size 000) moistened with homologous serum or
physiologic saline to which EDTA has been added. The tip
of the brush is rolled gently in the exposed marrow and several stripes of marrow are made on glass slides; multiple
slides may be prepared for special stains or techniques. For
dogs, imprints of sternal marrow or aspirates from the iliac
crest, sternum, proximal humerus, or trochanteric fossa of
the femur may be collected and placed in EDTA/physiologic
saline. When marrow granules have been obtained, crush
preparations may be prepared by attening and spreading the
marrow ecks between two glass slides to produce a smear
(Figure 11).
Preparations from samples obtained without EDTA may
be used as long as the smears are prepared immediately after sample collection. Smears can be made from antemortem
or postmortem samples. Postmortem collections should be
performed as soon as possible following sacrice (within
minutes); in the dog, it has been indicated that postmortem
samples should be taken within 30 minutes following the animals death (Tyler and Cowell, 1989). The air-dried smears
are then stained using a general procedure for Romanowskytype staining of blood lms. Since marrow smears tend to
be thicker than blood lms, staining times must be modied (at least 2x longer) to ensure adequate staining. Also,
formalin may affect the staining qualities of the marrow
smears, care should be used to avoid contact of the marrow or marrow smears with formalin or its vapors prior
to staining. Using the 100x objective, a morphological assessment of individual cell lineages and a differential bone
marrow count is performed. Differential counts are best performed on 500-cell counts but lower counts (e.g., 250-cell
counts) have been utilized. The differential should classify
cells by type (e.g., myeloid, erythroid, megakaryocytic, lymphoid, macrophage, etc.) and stage (e.g., rubriblast, prorubricyte, rubricyte, metarubricyte, etc.) (Figures 1217). Relative
percentages of the cells, M:E ratios and maturation indices
can then be calculated. Examples of differential counts of

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FIGURE 7.Low (A) and higher (B) magnication of the femoral marrow from a normal young adult dog. Photomicrograph courtesy of Drs. Hans Harleman and
Kathryn Bowenkamp.
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FIGURE 8.Low (A) and higher (B) magnication of the distal femoral marrow from a control female F344 rat at the end of a 90-day study. Adipocytes occupy
more of the marrow space than hematpoietic cells in this rat. There is a wide range of normal hematopoietic cellularity in bone marrow with high variability among
different bone sites. This degree of hematopoietic cellularity is within the normal range for a 4- to 5-month old F344 rat.

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FIGURE 9.This low (A) and higher (B) magnication of the distal femoral bone marrow is from a control female B6C3F1 mouse at the end of a 90-day study
and shows a difference in bone marrow hematopoietic cellularity in comparison to the control rat in Figure 8. As is the case in the rat, there is also a wide range of
normal hematopoietic cellularity in mouse bone marrow. Mice generally have a more cellular bone marrow than rats.

bone marrow from normal rats and dogs are presented in

Table 3.
For histopathology, numerous variables regarding specimen collection and processing (e.g., xation, decalcication,

embedding, sectioning and staining) may affect the quality of

the sample to be evaluated and must be considered. For a detailed discussion, the reader is referred to references regarding
processing procedures (Bennett et al., 1976; Beckstead et al.,

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FIGURE 10.Bone marrow from a normal 6-month-old 129 mouse. This highly cellular bone marrow has areas of myeloid (M) and erythroid (E) hematopoiesis
as well as megakaryocytes (arrows).

1981; Moosavi et al., 1981; Weiss, 1987; Wickramasinghe,

1992; Callis and Sterchi, 1998; Hedrich and Bullock, 2004;
Fero, 2005). A brief description follows.
Fixation
If collected at necropsy, marrow should be collected as
soon as possible for histological assessment. Fixative solutions stabilize tissue by cross-linking proteins and the degree
of cross-linking may result in denaturation of proteins and affect morphological, cytochemical or immunohistochemical
qualities of the specimen. Because its readily available, 10%
neutral buffered formalin (10% NBF) is the most commonly
used xative. For routine H&E sections, no special handling
is required prior to tissue processing. 10% NBF-xed tissues
may be used for frozen sections and evaluation of adipocytes.
For specimens to be used for immunohistochemistry, a prolonged xation in 10% NBF may be detrimental (e.g., loss
of antigenic epitopes due to continued amino group crosslinking and alteration of tertiary protein structure). Transferring the tissue to 70% ethanol after a 12-hour xation reduces
xation-related effects.

Zenkers-type solutions (e.g., Zenkers-acetic acid, B5,

Hellys) are an often recommended, but, not commonly used
xative for bone marrow sections. These xatives must be
made fresh prior to use and tissues must be washed prior
to processing or treated with Lugols iodine to remove pigments. Tissues are adequately xed in 12 hours but then
need to be transferred to alcohol; tissues become hard and
brittle with prolonged xation. Fine precipitates may form in
the tissue and may become problematic when special stains
are applied (e.g., silver stains). Additionally, the xative
constituent mercuric chloride is toxic and readily absorbed
through skin. There are zinc chloride-based, Zenkers-like xatives (e.g., AZF); these have similar properties to Zenkers
uids but no mercuric chloride. Zenkers-type xatives are
useful for immunohistochemical procedures. And, they act as
a mordant for the Giemsa stain resulting in improved nuclear
detail.
Bouins solution is the preferred xative by some investigators for bone marrow sections. Tissues are xed overnight,
followed by a 2 to 4-hour post-xation wash (in water) and
storage in 70% ethanol. Tissues become hard and brittle with

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TOXICOLOGIC PATHOLOGY

FIGURE 11.Bone marrow smear from a normal mouse. There is an even dispersal of hematopoietic cells admized with adipocytes and little blood contamination.
Note the size difference of the megakaryocytes (arrows) compared to the other hematopoietic cells.

prolonged xation. The picric acid constituent stains tissues

yellow. Since Bouins contains acetic acid, it may be used
for decalcication; this may take an extended period of time
and requires frequent (weekly) solution changes. Delicate
morphological detail is not well preserved, but it does act
as a mordant for basic aniline dyes (e.g., H&E) improving
staining.
Decalcication
For parafn-embedded sections, bone marrow specimens
must be decalcied prior to sectioning; specimens for plastic
embedding do not. There are numerous types of decalciers
(e.g., acids, chelators, resins) and methods (e.g., immersion,
sonication, microwave, ion-exchange) for the decalcication
of bone marrow specimens. If preservation of enzyme reactivity or antigenic sites is required, selection of the method
of decalcication is important. With larger specimens, some
damage to tissue morphology, due to decalcication, is almost
unavoidable (especially with the use of acid decalciers).

Both organic and mineral acids are commonly used for decalcication of bone marrow specimens. Organic acids (e.g.,
formic and acetic) decalcify slower than mineral acids (e.g.,
HCL and nitric). Mineral acids are commonly used in the
rapid-type decalcication methods. Overdecalcication of
tissue with acids, particularly mineral acids, results in tissue destruction. Thus, tissue in acid decalciers should not
be left unchecked for extended periods (e.g., over a weekend).
For proper decalcication, there must be even distribution of
acid around bone sample. Thus, tissue suspension or mild
agitation, such as gentle mixing (e.g., shaker/rocker) or air
bubble percolation, may be useful. Additionally, acid decalciers may need frequent solution changes (e.g., daily) for
adequate decalication. In general, acid decalcication is not
recommended for samples needed for enzyme- or immunostaining. This caveat is particularly appropriate for the mineral acids like HCL or nitric; the organic acids appear to
be somewhat better. All acid decalciers, especially mineral
acids, reduce morphological quality and Giemsa stains may
be unacceptable due to loss of basophilic-staining structures.

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561

FIGURE 12.In this bone marrow smear from a normal rat, a variety of cell types includes the following: BN = band neutrophil; L = lymphocyte; MF =
mitotic gure; MM = metamyelocyte; MR = metarubricyte; R = rubricyte; RM = ring form myelocyte; SN = segmented neutrophil. 13.In this bone marrow
smear from a normal rat, multiple cell types include: BN = band neutrophil; MM = metamyelocyte; MR = metarubricyte; R = rubricyte; RM = ring form
myelocyte; SN = segmented neutrophil. 14.Bone marrow smear from a normal rat. Mast cells (MC) are present in an admixture of mostly myeloid precursors.
15.Bone marrow smear from a normal rat. A multinucleated megakaryocyte is surrounded by eosinophilic myeloid precursors (E) and other hematopoietic
cells.

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TRAVLOS

FIGURE 16.Bone marrow smear from a normal mouse. A macrophage (Mac) with phagocytized debris/pigment is surrounded by a prorubricyte (PR), a segmented
neutrophil (SN), and a band neutrophil (BN). A rubricyte (R) is also present. 17.Bone marrow smear from a normal rat. A cluster of large adipocytes (center) is
surrounded by an admixture of hematopoietic cells.

Chelating agents, such as EDTA, are also commonly used

for decalcication of bone marrow specimens. Decalcication proceeds at a slower rate compared to the acid decalciers, especially compared to mineral acids. EDTA decalcication is recommended for enzyme- and immuno-staining

procedures. The action of EDTA is pH dependent (i.e., the

higher the pH, the faster the decalcication). Since a high alkaline pH may also cause tissue destruction and resultant
loss of cytological, enzyme or immunostaining qualities,
however, the use of EDTA at a high pH (e.g., pH 10)

TABLE 3.Examples of bone marrow differential counts for the adult dog and rata .
Dog
Species Source

Myeloid Series
Myeloblast
Promeylocyte
Myelocyte (neutrophilic)
Myelocyte (eosinophilic)
Metamyelocyte (neutrophilic)
Metamyelocyte (eosinophilic)
Band (neutrophilic)
Band (eosinophilic)
Segmenter (neutrophilic)
Segmenter (eosinophilic)
Basophils
Total Myeloid Series
Erythroid Series
Rubriblast
Prorubricyte
Rubricyte
Metarubricyte
Total Erythroid Series
Myeloid:Erythroid Ratio
Lymphocytes
Plasma cells

Rat

Melveger et al. (1969)

Jain (1986e)

Kiczak et al. (1976)

0.9 0.2
2.1 0.4
6.3 1.0
0.6 0.2
7.9 2.1
0.7 0.3
11.3 2.2
1.2 0.4
23.5 1.3
0.8 0.5
0.02 0.04
55.3

0.0
1.3
9.0
0.0
7.5
2.4
13.6
0.9
18.4
0.3
0.0
53.4

5.8 0.2
5.5 0.2

6.2 0.3

11.0 0.4

12.4 1.1
4.0 1.3
0.2 0.04
45.1

1.0 0.9
2.0 0.9
4.4 0.9

6.8 1.4

14.8 2.7

11.9 3.3
2.7 0.9
0.5 0.03
44.1

6.5 0.5b

27.6 4.4c
34.1
1.7 0.4
8.2 2.7
0.7 0.3

0.2
3.9
27.0
15.3
46.4
1.15
0.2

8.8 0.8d

21.4 0.2e
30.2
1.49
0.5 0.03
3.5 0.03

0.3 0.5
1.7 0.8
20.6 1.7 f
13.2 2.0
35.8
1.23
18.7 3.6
0.4 0.5

Values for cell types are presented percentages either as a value or mean SD.
Value includes rubriblasts and prorubricytes.
Value includes rubricytes and metarubricytes.
d
Value includes rubriblasts, prorubricytes and basophilic rubricytes.
e
Value includes and polychromatophilic rubricytes and metarubricytes.
f
Value includes and basophilic, polychromatophilic and normochromic rubricytes.

b
c

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Valli et al. (2002)

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FIGURE 18.Comparison of details of cytomorphological features between a 5-micron (A) and a 3-micron (B) section stained with hematoxylin & eosin. Femoral
marrow from normal B6C3F1 mice.

must be avoided. With initial placement of tissue in a sufcient amount of chelator, solution replacement is usually
not needed.
These decalciers may be applied individually or in combination using immersion, microwave, sonication or electrolytic methods. For immersion techniques, tissues are
placed in adequate decalcier at ambient temperature. This
is the slowest of the aforementioned methods but causes the
least artifactual tissue damage if the sample is overdecalcied and H&E staining is generally adequate. Microwave
techniques utilize a microwave to heat a water bath in which
a container with tissue immersed in a decalcier is placed.
Decalcication is enhanced (especially with the mineral
acids) but it is easy to heat-damage the tissue (especially at
>45 C). Using 70% power for 20 minutes with 10-minute
cool-down intervals help diminish the effects of heat. H&E
staining is generally adequate, but one may see dark marrow
components with sparse, smudged nuclei (probably related
to heat damage). Sonication techniques involve immersing
the tissues in a sonicator containing decalcier and sonicating the tissues. The speed of decalcication is enhanced and

H&E staining is adequate, but there can be cytological alterations similar to what occurs using the microwave method.
The electrolytic method involves suspending a single tissue
in an acid decalcier between 2 electrodes and passing a
weak electrical current to enhance decalcication time. This
method is slightly faster than immersion and the staining is
comparable to the immersion method. However, the setup is
elaborate and not amenable to a high-throughput operation;
this requires frequent solution changes and heat damage can
occur.
Decalcication by ion-exchange varies from the above
methods in that a calcium-sequestering resin is used in combination with an acid decalcier. This technique appears to
be faster than immersion methods and appears to provide the
best morphology for tissues stained with H&E. A resin (e.g.,
Win-3000) is placed in the bottom of a container, which is
then lled with an acid decalcier (typically an organic acid
such as formic acid); the tissue(s) are immersed in the decalcier. Since the resin sequesters the calcium, a chemical precipitation method for determination of demineralization endpoint cannot be performed. The resin may be reused and daily

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TRAVLOS

solution changes are unnecessary. However, this method is

not amenable to high throughput operations due to the availability and cost of the resin, limited tissue capacity in the
decalcication chamber and the time required for thorough
washing of the resin for reuse.
Demineralization Determination
Determination of the demineralization endpoint is an important step. Overdecalcication results in tissue destruction
and underdecalcication results in poor dehydration and inltration and, ultimately, sectioning. The X-ray method is the
most accurate but requires the appropriate equipment. Also,
tissues treated with xatives containing mercury or other
heavy metals are rendered radiopaque; thus, X-ray cannot
be used for these tissues. The chemical precipitation method
(e.g., calcium oxalate precipitation) is reliable and easily applied for acid decalciers. A modication of this method also
may be used for EDTA decalcied tissues. Mechanical bending or probing is a subjective assessment and is by far the
easiest method and is often done. But, it is not recommended
as it is not reliable and the tissue manipulation may disrupt
architecture or dislodge soft tissue components from the sample. Cutting the sample has also been used and appears to be
an accepted practice.
Tissue Processing and Staining
Because of its routine use, H&E-stained sections of
parafn-embedded bone marrow tissue are typically evaluated histologically. However, H&E does not provide consistent hematopoietic cell differentiation. Section thickness is
an important factor and 3-micron sections provide better cellular morphological detail compared to thicker (5-micron)
sections (Figure 18). Cytological quality is not, however, as
good compared to Romanowsky-stained cytology preparations. Giemsastained sections provides better morphological detail compared to H&E-stained sections and are more
easily compared to Romanowsky-stained smears.
For the Giemsa stain, however, acid-decalcied tissue results in loss of basophilic-staining structures. The use of
chelating-type decalciers improves Giemsa staining. Plastic embedding provides a signicant improvement in cellular
morphology. Additionally, there is no need for decalcication, thus, reducing shrinkage artifact and loss of cellular detail related to decalcication methods. Thin sections (3 microns) can be easily produced, improving cytological quality.
However, plastic embedding is time, labor and cost intensive
and, thus, not justied for a high-throughput operation. Prussian Blue stain is used for the assessment of iron stores; acids
in decalciers or xatives may washout iron stores. Thus,
tissue iron could be underestimated.
SUMMARY
Since hematopoietic system is a potential target organ of
chemical exposure, evaluation of the blood and bone marrow is important component of any toxicity or safety assessment study. Evaluation of the hematopoietic system should
routinely include a CBC and differential and bone marrow
histopathology. While the CBC provides information regarding possible compound-related effects demonstrated in the
peripheral blood, morphological evaluation of bone marrow
provides information about bone marrow tissue architecture

TOXICOLOGIC PATHOLOGY

(e.g., cellularity, cell linages, vascular or stromal alterations,

inammation, necrosis), estimation of iron stores and identication of other features (e.g., pigment, infectious agents,
proliferative or neoplastic disorders) that otherwise would be
missed by examination of peripheral blood alone. The quality
of the marrow sections, however, is governed by numerous
variables related to specimen collection and processing and
must be considered.
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