International Journal of Laboratory Hematology

The Official journal of the International Society for Laboratory Hematology

REVIEW

INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY

Bone marrow evaluation for pediatric patients

M. PROYTCHEVA

Department of Pathology,
College of Medicine, University
of Arizona, Tucson, AZ, USA
Correspondence:
Dr Maria Proytcheva, Department of Pathology, College of
Medicine, University of Arizona,
1501 N. Campbell Avenue, Tucson, AZ 85724, USA.
E-mail: mproytcheva@
pathology.arizona.edu
doi:10.1111/ijlh.12073

S U M M A RY

Due to the immaturity of the hematopoietic system at birth and

different oxygenation and immune response needs of the growing
organism, the bone marrow composition at birth and early infancy
differs as compared to older children and adults. These age-related
differences, while generally recognized, are not well known to the
world of hematopathology. The purpose of this article is to address
the current limitation of the literature by reviewing the bone marrow ontology, its composition at birth, and the changes occurring
during early infancy, and to compare these findings to adults. The
review also provides a useful framework for bone marrow examination in children.

Received 25 January 2013;

accepted for publication 14
February 2013
Keywords
Bone marrow, hematopoiesis,
normal, neonatal, pediatric

INTRODUCTION
The bone marrow (BM) is the last blood-forming tissue
that develops in ontogenesis and from birth, and thereafter, it is the major hematopoietic site. It is a functionally dynamic organ, and its composition depends
highly on the needs for oxygenation, fighting infections, and proper hemostasis. As such needs vary drastically during different stages of development as well
as early childhood and later in life, the BM composition also changes to meet those needs. Therefore, it is
important when evaluating BM of a child to distinguish between the findings due to the normal development and those that result from various diseases.
The hematopoiesis in the bone marrow begins in
the long bones at 68.5 weeks of gestation and is
completed by 16 weeks of gestation with final organi 2013 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2013, 35, 283289

zation of the bones into areas of dense hematopoiesis

surrounded by areas of fully calcified bone [1, 2].
Between the 11th and 24th weeks of gestation, both
the liver and BM are hematopoietic organs concomitantly, yet each supports a different set of hematopoietic lineages: the erythropoiesis occurs mostly in the
fetal liver and the granulopoiesis and lymphopoiesis
mostly in the BM. The total marrow volume markedly
increases during the second trimester, and after the
24th week of gestation, the hematopoiesis shifts from
the fetal liver to the BM.
From birth onward, the BM is the major hematopoietic site. At birth, all cavities of the skeleton contain red hematopoietic BM and almost no fat. In the
first year of life, the hematopoiesis occurs in both the
axial and appendicular skeleton, and thereafter, there
is a gradual decrease in the hematopoiesis in the long
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bones until about age 15. At that age, active hematopoiesis is confined to the proximal quarters of the
shafts of the femur, humerus, and the axial skeleton
[3].
The BM is a functionally dynamic structure, and if
the needs for erythrocyte, leukocyte, or platelet production increase, the hematopoiesis expands, and the
fat is replaced by bone marrow. In young children,
however, an increase in hematopoiesis is accommodated by a reduction in the proportion of marrow
sinusoids, and in severe congenital anemia, the
marrow cavities expand leading to bone deformity.
The BM is located between the bone trabeculae
and has a highly complex three-dimensional structure
composed of extracellular matrix, stromal cells including osteoblasts, and capillary venous sinuses. The
localization of the various hematopoietic elements is
nonrandom, and in histologic sections, the cells with
proliferative activity are preferentially located near the
bone trabeculae, and the differentiated elements are
observed in the central, intertrabecular spaces [4].
The early myeloid progenitors are localized in the
paratrabecular areas close to the adventitia of the
small arteries. Normally, the layer of immature granulocytes does not exceed 23 rows of maturing cells.
With maturation, the cells migrate to the intertrabecular spaces.
The erythroid progenitors mature and differentiate
in erythroblastic islands that consist of a central macrophage a key component of the erythroid differentiation surrounded by developing erythroblasts. As
the erythroblasts become more differentiated, the erythroid islands migrate toward sinusoids because they
are a mobile structure. The erythroid islands are not
readily observable on histologic section, but can be
seen on bone marrow aspirates, particularly in
patients with erythroid hyperplasia.
Megakaryocytes reside adjacent to marrow sinusoids, which allow easy shedding of platelets directly
into the circulation.

AG E - S P E C I F I C D I F F E R E N C E S I N T H E B M
Due to the immaturity of the hematopoietic system at
birth and the nature of hematopoiesis with its dynamic
response to the oxygenation needs and immune
response of the growing organism, several important differences between the BM cellularity and composition in

children and adults exist (Table 1) [58]. At birth, the

bone marrow is almost devoid of fat (100%) and contains only hematopoietic elements, and with advance of
age, the red hematopoietic marrow is replaced by fat.
The BM cellularity in normal infants 3 months of age
or younger is 80% or more. Particles obtained from
the iliac crest and the sternum have compatible cellularity and, in normal children aged 18 months to 11 years,
ranged between 50% and 70% [7].
In a study of 448 healthy BM donors and children
with non-neoplastic hematologic disorders or nonhematologic malignancies, Friebert et al. [6] found that
children younger than 2 years have the highest BM
cellularity (79.8  15.7%) and with age the cellularity
declined to 68.6% (16.5%) for children aged
24 years, to 59.1% (20.1%) for children aged 5
9 years, and to 60% (17.9%) for children aged 10
14 years. Thus, BM cellularity of 60% is achieved
during the first 5 years of life and remains relatively
constant compatible with other age groups after that.
Similar findings are obtained using imaging studies.
Ogawa et al. [9] found 60.0  20.0% cellularity in the
BM of children aged 09 years declining to
56.5  4.4% for ages 1019 years. These findings
clearly demonstrate the inaccuracy of the concept of
BM cellularity determined by the formula 100%
minus the age of the patient. Using such an approach
will significantly overestimate the BM cellularity in
young children and underestimate the cellularity in
older adults. Thus, it should not be applied.
Similarly to the BM cellularity, the composition of
the marrow is also age dependent [5, 1013]. In a prospective study of BM composition of 88 clinically
healthy children with normal peripheral blood counts,
serum proteins, and transferrin saturation of at least
16%, Sturgeon found that at birth, the BM has a predominance of myeloid progenitors and relatively low
number of erythroid progenitors and lymphocytes. The
most significant changes take place during the first
month of life and are manifested by a decrease in the
percentage of myeloid progenitors and erythroblasts
and an increase in the number of lymphocytes
(Figure 1). The total myeloid component initially
decreases during the first 2 weeks of life followed by a
sharp drop around the 3rd week to reach a steady level
around 3035% after the 1st month. The marrow
eosinophils range between 2 and 3%, the monocytes
remain below 2%, and the basophils remain below 1%
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M. PROYTCHEVA | BM EVALUATION IN CHILDREN

285

Table 1. Bone marrow cellularity and cellular composition in children of various ages
Age

Cellularity Cellular composition

Newborn

90100%

Neonate (birth
to 28 days)

90%

Infant (1 month 8090%

to 1 year)

25 years

6080%

612 years

5070%

Older than
12 years and
adults

4060%

Myeloid hyperplasia with a shift to immaturity.

Less than 5% blasts
Decrease in the number of myeloid cells in the first 2 weeks
of life
Decrease in the number of erythroid progenitors
Megakaryopoiesis:
Monolobated small megakaryocytes
Gradual increase in the number of lymphoid cells, mostly
B cells
M:E ratio 512 : 1*
Myelopoiesis:
Reaches a steady state level ~3035%
Erythropoiesis:
After the initial drop, a transient peak in the 2nd month,
there is a second decrease at 34 months with subsequent
stabilization of the total erythroblast count of 79%;
Relative erythroid hypoplasia is most pronounced during
the physiologic nadir
Megakaryopoiesis:
Monolobated small megakaryocytes
Diffuse interstitial lymphocytosis
Lymphoid cells, the major population after the 1st month
(47.2  9.2%)
Predominance of normal B cell progenitors (hematogones)
T-cells and NK-cells minor components
Lymphoid aggregates not normally present
Plasma cells rare, polytypic, frequently associated with
various diseases
L:M:E ratio ~ 6 : 5 : 1*
Iron stores absent in young children
M:E ratio 34 : 1
Increase in the myeloid and erythroid component and
decrease in the number of B cells and hematogones and
slight increase in the number of T cells. Note, an increased
number of hematogones can be seen in infections or
children with congenital cytopenias due to primary bone
marrow failure.
Detectable stainable iron after age 45 years
M:E ratio 34 : 1
Less than 20% lymphocytes
T cells exceed B cells
Iron stores reach adult level
M:E ratio 34 : 1
Less than 20% lymphocytes
T cells exceed B cells
Lymphogranuloma and lymphoid aggregates may be
present, their number increases with age

Bone trabeculae

Very active bone

remodeling
Incomplete ossification
Prominent osteoblastic
rimming

Prominent bone
remodeling

Bone remodeling
may be evident,
particularly boys
Inconspicuous osteoblasts
and osteoclasts
No bone remodeling

*M:E, Myeloid to erythroid ratio; L:M:E, the relative proportions of lymphocytes, myeloid, and erythroid progenitor.
Based on data from Rosse, C. et al. Bone marrow cell populations of normal infants: the predominance of lymphocytes. J Lab Clin Med 1977; 89:122540.

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(a)

(b)

(c)

(d)

Figure 1. Bone marrow studies from a 4-month-old infant. (a) Bone marrow aspirate smear demonstrating
hematopoietic progenitors along with numerous mature and immature lymphoid cells (WrightGiemsa stain).
(bd) Multiparameter flow cytometry showing a significant population of CD19+/CD10+/CD38+ B cells with a
variable expression of CD20, a mature B-cell marker. Note, the CD20+ mature B lymphocytes comprise a large
proportion of all B cells. A small number of T cells is also present (not shown). This flow cytometric pattern is
consistent with normal B cell progenitors, hematogones.

at all times. The plasma cells and other marrow cells

comprise only a small fraction of the cellularity.
The number of erythroid progenitors also decreases
significantly during the first month of life and after a
transient peak at the end of the second month is followed by more gradual but significant secondary
decrease through months 3 and 4 to stabilize and
account for 79% of total erythroblasts at various
stages of maturation. These changes are broadly
followed by the peripheral blood reticulocyte count.
The number of megakaryocytes in children is compatible with adults. However, there is a difference in
their size and lobulation. Small megakaryocytes with
uniform size and monolobated nucleus, occasionally
forming clusters, are characteristic for young children
and should not be mistaken for abnormal megakaryopoiesis. In a study of the size of megakaryocyte of 61
young children, Fuchs et al. [14] found a single peak sig-

nifying uniform small size at the youngest ages, which

diverges into separate clusters of smaller and larger cells
beginning at 2 years that is followed by an overall shift
toward larger megakaryocytes at age 4 years.
Another significant difference between the BM composition of infants and older children and adults is the presence of a high number of lymphocytes that increase
significantly in the immediate neonatal period to become
the largest population in the marrow (47.2  9.2%) by
the end of the first month [11]. During the next
17 months, the number of lymphocytes is relatively stable
whereupon it begins to decrease gradually. During the first
4 years of life, the B cells comprise 65% of the lymphocytes in contrast to adult marrow where T lymphocytes
predominate [12]. Most of the B cells, 80%, are normal
B-cell progenitors, hematogones. These cells comprise a
heterogeneous population of immature, surface immunoglobulin-negative B cells that include early, intermediate,
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M. PROYTCHEVA | BM EVALUATION IN CHILDREN

and late forms (Figure 1). The early, stage I, hematogones

express CD34 and TdT and are mostly CD20 negative. The
stage II and stage III hematogones loose CD34 and TdT
and gradually acquire CD20. Furthermore, stage III hematogones acquire cytoplasmic l and loose CD10. During the
first 2 years, the CD20 and surface immunoglobulin-positive nave B cells comprise a minor population, and with
age, their number gradually increases, whereas the number of hematogones decreases. After the age of 4 years, the
overall number of B cells gradually decreases, and this
decline is accompanied by an increase in the number of
CD3+ T lymphocytes. These T cells are mature and express
either CD4 or CD8, and the percent of CD8+ cells is at least
twice the percentage of CD4+ cells. The number of NK cells
in the marrow is low and does not depend on age.
The proliferative capacity of BM in children and
young adults determined by Ki-67 appears to be higher
and the apoptotic rate lower as compared to older people [9]. Active bone remodeling, prominent osteoblastic
rimming, osteoid seams, and incomplete ossification are
frequent in children and do not signify pathology.

B O N E M A R R OW E X A M I N AT I O N I N C H I L D R E N
The bone marrow diagnosis in children as well as in
adults is based on the integration of data from various
diagnostic studies, including peripheral blood count and
film evaluation, BM aspirate smears, particle clot sections, BM trephine biopsy, and imprint morphology
along with the results of cytochemistry, immunophenotypic analysis, cytogenetics, and molecular studies [15].
The most frequent indication for bone marrow examination in children includes investigation of abnormal blood
counts suggestive of BM pathology; initial workup for a
child with peripheral cytopenias and suspected primary
bone marrow failure or occult malignancy; unexplained
organomegaly in children with mass lesions inaccessible
for biopsy; following response to therapy for acute
leukemia and detection of minimal residual leukemia; to
determine BM engraftment following a stem cell
transplant; and staging for Hodgkin or non-Hodgkin
lymphoma, neuroblastoma, or rhabdomyosarcoma. Of
note, unlike neuroblastoma and rhabdomyosarcoma
that metastasize to the BM frequently, other small blue
cell tumors such as the Ewing sarcoma family of tumors,
Wilms tumor, and nonrhabdomyosarcoma soft tissue
sarcomas rarely involve the BM; thus, BM examination
is not part of the routine staging for those tumors.
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287

In young children, BM studies are not indicated for

the determination of iron stores, as stainable iron is
absent from the marrow during the first year of life
and storage iron progressively increases and reaches
adult levels only by the fifth to 6th year [16]. Similarly, BM studies are not required in the initial evaluation of children with thrombocytopenia prior to
initiation of steroid therapy as such studies have been
shown to contribute little and not to change significantly the quality-of-life years of such children.
In children with suspected acute leukemia, the diagnosis can be established on BM aspirate and peripheral
blood alone, and BM biopsy may not be necessary. However, in children with suspected aplastic anemia, BM
involvement by small blue cell tumor, or staging for
Hodgkin or non-Hodgkin lymphoma, a BM biopsy will
be necessary to establish the diagnosis. In such studies,
sampling both, the BM aspirates and biopsy, will
enhance the chances of arriving to the right diagnosis.
The iliac crest is the most frequent BM sampling site
in children. In newborns and neonates, conventional
BM biopsies may be difficult to perform, so alternative
techniques, such as obtaining marrow clot sections,
can be successfully utilized [17, 18]. The BM particles
in clot sections contain preserved marrow architecture,
so that they provide information on the overall BM
cellularity and the number and morphology of megakaryocytes. Such sections can also be used for immunohistochemical stains or other special stains. While the
adult requirement for the size of BM biopsy of at
least 1.52 cm may not be achievable in very young
children, an effort should be made so that sufficient
material is provided for proper examination [15].
After chemotherapy, BM aspirates may be paucicellular and may not contain hematopoietic elements. In
the absence of particles or megakaryocytes, or other
hematopoietic precursors, the sample should be
reported as dry tap or peripheral blood [15]. In
such instances, fresh BM biopsy is suitable for flow
cytometry that will provide valuable information on
the presence or absence of minimal residual disease.
Examination of BM aspirate smears at low-power
magnification is essential for determining the number
and cellularity of marrow particles, megakaryocytes as
well as presence of tumor clumps or abnormal cells that
may be of low incidence. This is particularly important
in paucicellular BM smears in children with suspected
neuroblastoma or rhabdomyosarcoma.

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M. PROYTCHEVA | BM EVALUATION IN CHILDREN

Higher magnification provides valuable information

on the composition of marrow particles, cytological
details, and cell inclusions. As with adults, both quantitative and qualitative evaluation of all cell lineages is
important in generating a differential diagnosis. The
myeloid-to-erythroid ratio should also be documented. The BM trephine biopsies provide valuable
information on the overall cellularity, abnormal localization of progenitors, fibrosis, and presence of
extrinsic cells or lymphoid aggregates. In children,
lymphoid aggregates are rare and often present in
individuals with underlying immune deficiency, autoimmune disorders, or other systemic diseases.
The adult guidelines for BM reporting are applicable
to children [15, 19]. The BM report should include BM
cellularity described as acellular, reduced, normal,
increased, or markedly increased along with quantitative
and qualitative comments on all cell lineages myeloid
and erythroid progenitors with M:E ratio, megakaryocytes, lymphocytes, plasma cells and any abnormal
cells such as cells extrinsic to the bone marrow, leukemic blasts, increased number or abnormal mast cells,
and the presence of histiocytes displaying hemophagocytosis. It is important to note that histiocytes displaying
hemophagocytosis is a nonspecific finding, and while in
proper clinical settings, it may be indicative of hemophagocytic lymphohistiocytosis, it may also be seen in a

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AC K N OW L E D G E M E N T
The author thanks Dr Deborah Fuchs for the critical
reading and comments of the manuscript.

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