Cytometry Part B (Clinical Cytometry) 60B:1–13 (2004)

Improving Lab Practice

Immunophenotypic Differentiation Patterns of Normal
Hematopoiesis in Human Bone Marrow:
Reference Patterns for Age-Related Changes
and Disease-Induced Shifts
E.G. van Lochem,* V.H.J. van der Velden, H.K. Wind, J.G. te Marvelde,
N.A.C. Westerdaal, and J.J.M. van Dongen
Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

Background: The abundance of monoclonal antibodies (mAb) and the routine use of quadruple stainings
in flow cytometry allow stepwise analysis of bone marrow (BM) samples that are suspected for abnormal
hematopoiesis. A screening phase that precedes lineage-specific classification phases should be sufficient
to assess whether the BM has a normal or abnormal composition, as well as to identify the abnormal
differentiation lineage.
Methods: For a quick and easy flow cytometric screening of BM samples, we selected six quadruple
immunostainings that cover multiple differentiation stages of the B-cell, monocytic, granulocytic, and erythroid
lineages: TdT/CD20/CD19/CD10 and CD45/CD34/CD19/CD22 for B cells, CD34/CD117/CD45/CD13.33 for precur-
sor granulocytic and precursor monocytic cells (myelo/monoblasts), CD14/CD33/CD45/CD34 for monocytic cells,
CD16/CD13/CD45/CD11b for granulocytic cells, and CD71/CD235a/CD45/CD117 for erythroid cells.
Results: The six quadruple immunostainings reveal specific staining patterns in normal BM, which allow
the recognition of various subpopulations of the respective lineages. These staining patterns can be used as
a frame of reference for recognition of normal and abnormal BM development. Examples of normal
(age-related) variations in these otherwise stable staining patterns are presented together with several
abnormal differentiation patterns.
Conclusions: Although alternative immunostainings can be used (e.g., including NK- and T-cell markers),
we feel that the selected six stainings represent a comprehensive and easy screening phase for quick
identification of shifts in the composition of the studied differentiation lineages, reflecting age-related
changes or disease-induced BM abnormalities. © 2004 Wiley-Liss, Inc.

Key terms: flow cytometry; normal bone marrow; staining patterns; immunophenotyping; expression profile

Flow cytometric analysis of bone marrow (BM) samples
is performed routinely for identification, frequency assess-
ment, and further characterization of the various leuko-
cyte subpopulations when abnormal hematopoiesis is sus-
pected. The BM compartment is a complex tissue
containing cells of multiple hematopoietic lineages with
various maturational stages per lineage. In healthy individ-
uals, the BM precursor cells guarantee continuous produc-
tion of the various hematopoietic differentiation lineages.
The differentiation and maturation can be monitored by
changes in cytomorphology and immunophenotype. Es-
pecially in the B-cell lineage, multiple stages have been
defined based on their immunophenotype (1–5). Also in
the monocytic, granulocytic, erythroid and thrombocytic
lineages several differentiation stages can be identified by
immunophenotyping (6 – 8). Knowledge of the expres-
sion (levels) of various lineage-specific, immature, and
mature markers in normal hematopoietic development
provides a frame of reference for recognition of abnormal
differentiation patterns.
Abnormal BM hematopoiesis may result from (1) an
arrest during precursor B-cell differentiation; (2) BM re-
generation after immunosuppressive therapy or as a re-
sponse to infection; (3) dysplasia in one or more myeloid
lineages; (4) increased turnover of hematopoietic cells
*Correspondence to: E.G. van Lochem, Department of Immunology,
Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein
40, 3015 DR Rotterdam, The Netherlands.
E-mail: e.vanlochem@erasmusmc.nl
Received 18 August 2003; Accepted 29 October 2003
Published online 14 June 2004 in Wiley InterScience (www.
interscience.wiley.com).
DOI: 10.1002/cyto.b.20008

© 2004 Wiley-Liss, Inc.

2 VAN LOCHEM ET AL.

caused by peripheral cell depletion; or (5) unregulated Table 1

expansion of neoplastic hematopoietic cells. Most of MAbs Used for the Six Selected Quadruple Immunostainings
That Cover the Development of the Four Major Hematopoietic
these abnormalities will present as major shifts in BM Lineages in Normal BM
composition or as specific shifts in the relative distribution
of subsets in one or more lineages. In the case of a B-cell CD Clone Fluorochromes Manufacturer
differentiation arrest, the absence of the more mature B TdT — FITC SuperTechs
cells and the relative increase of precursor B cells will be CD10 HI 10a APC BD Biosciences
the most striking observation (5). In regenerating BM after CD19 4G7 PE BD Biosciences
SJ25C1 PerCP-Cy5.5 BD Biosciences
cytotoxic treatment, the immature precursor B cells will CD20 L27 PE BD Biosciences
also be overrepresented, but mature B cells will still be L27 PerCP BD Biosciences
detectable (9,10). In most BM-derived hematopoietic ma- CD22 S-HCL-1 APC BD Biosciences
lignancies, such as precursor B-acute lymphoblastic leuke- CD34 8G12 FITC BD Biosciences
mia (ALL) and acute myeloid leukemia (AML), the rela- 8G12 PE BD Biosciences
8G12 APC BD Biosciences
tively homogeneous leukemic cell population dominates CD45 2D1 FITC BD Biosciences
the BM compartment with profound suppression of other 2D1 PerCP BD Biosciences
hematopoietic lineages. These leukemic cells frequently CD117 104D2 PE BD Biosciences
have an aberrant immunophenotype not found in normal 104D2 APC BD Biosciences
CD11b D12 APC BD Biosciences
BM (11–16). Knowledge of the normal immunopheno- CD13 My7 RD1 Beckman Coulter
typic differentiation patterns in BM facilitates recognition WM15 APC BD Biosciences
of ALL and AML cells, even if they occur at low frequen- CD14 MOP9 FITC BD Biosciences
cies (17–20). CD16 5D2 FITC BD Biosciences
Studies of normal BM hematopoiesis are improved by CD33 906 RD1 Beckman-Coulter
P67.6 APC BD Biosciences
technological innovation of flow cytometry to the routine CD71 LO1.1 FITC BD Biosciences
use of four-color analyses. To generate significant data CD235a GPA PE Dako Cytomation
from these multi-parameter analyses, it is important to
MAbs, monoclonal antibodies; BM, bone marrow.
attune the composition of the monoclonal antibody
(mAb) combinations to the part of hematopoiesis under
study: the relative distribution of the BM leukocytes over
the major lineages; immature precursor cells; more ma- specific immunoglobulin subclasses or by using F(ab)2
ture differentiation stages; a specific lineage (B- or myeloid fragments, thereby avoiding interaction with FcR’s (24).
cells); or, a specific differentiation stage within a lineage. Third, most mAb are available with different fluoro-
Numerous leukocyte antigens have been studied exten- chrome-conjugates; the choice of fluorochrome-conjugate
sively for their expression (levels) on various hematopoi- should depend on the expression level of the antigen.
etic lineages and differentiation (11,21,22). It is not our Because of their strong fluorescence signal, PE- and APC-
aim to review the complete expression profiles of all conjugated mAb are the best choice for the detection of
leukocyte markers or to describe all minor BM subpopu- weakly expressed antigens, as well as for antigens with
lations. We focus on a limited set of six four-color immu- variable expression from weak to strong.
nostainings, which were selected for rapid screening of all The mAb clones and fluorochrome-conjugates that
major leukocyte (sub)populations in BM in order to easily were used for the six four-color immunostainings to assess
identify shifts or aberrancies in normal BM hematopoiesis the normal BM differentiation patterns are summarized in
(i.e., B-cell, myeloid, and erythroid lineages). We did not Table 1. Our standard immunophenotyping procedure
include immunostainings for identification of the natural uses ammonium chloride-lysed whole BM samples to pre-
killer (NK)-/T-cell lineage, since NK-/T-cell differentiation vent selective loss of cells and to conserve light scatter
does not take place in BM. characteristics (25). Cells were permeabilized for intracel-
lular immunostaining of TdT by using FACSBrand lysing
METHODOLOGY solution (BD Biosciences; San Diego, CA) (26). Data were
Technical Considerations for the Design of the acquired on a FACSCalibur (BD Biosciences) using Cell
Four-Color Immunostainings Quest Pro Software and analysed using both Cell Quest
The selection of the most informative mAb combina- Pro and Paint a Gate Pro software.
tions should preferably be based on the properties of
antibodies, antigens, and fluorochromes. First, several Strategy for the Design of Four-Color Immunostainings
clones in the same cluster of differentiation (CD) recog- In this report we present a limited set of six immuno-
nize different epitopes or the same epitopes with different stainings that unravel the normal differentiation patterns
affinity (22). For example, CD15 mAb can recognize sia- within the four major hematopoietic lineages in BM. Be-
lylated or nonsialylated epitopes, and CD34 antibodies cause T-cell development normally does not occur in BM,
(type I, II, and III mAb) recognize different epitopes with no T- or NK-cell-specific immunostainings were included.
variable sensitivity for enzymes (23). Second, aspecific In five of the six selected immunostainings, a lineage-
Fc-receptor (FcR) binding, causing an increased back- specific marker is used to select the lineage of interest.
ground signal, can be minimized by selecting mAbs of The combination of CD19 or CD13, with side-scatter (SSC)
 IMMUNOPHENOTYPIC REFERENCE PATTERNS OF NORMAL BONE MARROW
characteristics in a dot plot is useful to select and study
the immunophenotypic differentiation patterns within the
B cell and granulocytic/monocytic lineage, respectively.
Apart from lineage-specific markers and immature or ma-
ture markers that are restricted to a particular cell lineage,
markers with a much broader expression pattern can be
useful in the study of normal differentiation patterns in
BM. The pan-leukocyte marker CD45 gives additional in-
formation about the major subpopulations, as the CD45
antigen is differently expressed in various lineages and
differentiation stages (6,27,28). CD45 expression is high
on lymphocytes and monocytes, whereas granulocytes,
precursor B cells, precursor granulocytic cells, and pro-
erythroblasts are also CD45 positive, but at lower levels
(29,30). In contrast, (more) mature erythroid cells are
generally CD45 negative (6,31). Therefore, when com-
bined with SSC characteristics, CD45 can help distinguish
the major leukocyte (sub)populations in BM. CD34 is
another non-lineage-restricted marker, expressed by the
precursor stages of the various cell lineages in BM (32,33).
The normal differentiation patterns within a specific lin-
eage can be displayed by the application of “dynamic”
markers, i.e., markers that are gradually up- or downregu-
lated during differentiation. The immunophenotypic
changes are best illustrated when mAb directed against
markers with overlapping expression profiles are com-
bined in one staining. For example, CD10 and CD20 (in
B-cell development) and CD13 and CD16 (in myeloid
development) are informative combinations (4,7). Ideally,
the selected antibody combinations cover multiple con-
secutive differentiation stages, resulting in dot plots that
provide direct insight in the completeness and relative
distribution of the major subpopulations in the differenti-
ation lineage under study.
The composition of the selected immunostainings was
based on our extensive experience obtained during the
last decade with 900 –1,200 BM samples analysed each
year. All flow cytometric analyses of BM samples were
performed routinely with three-color immunostainings
during 1996 –1999 and with four-color immunostainings
from 1999 until the present.
We selected a set of six four-color immunostainings for
the analysis of the B-cell, monocytic, granulocytic and
erythroid cell lineages. Per lineage, the major subpopula-
tions are described and illustrated in the respective plots
and differentiation schemes. Normal age-related variations
in the expression profiles are discussed followed by ex-
amples of disease-induced shifts. We also discuss some
alternative markers or four-color immunostainings that
can be used for the same purpose.
BONE MARROW DIFFERENTIATION PATTERNS
B-Cell Differentiation
Normal B-cell differentiation. Early B-cell differenti-
ation has been extensively studied in BM using flow cyto-
metric immunophenotyping. Initially, most studies fo-
cused on precursor B-acute lymphoblastic leukemia
(precursor B-ALL) to unravel early B-cell development.
Later, studies on the B-cell compartment in normal BM
3
further unraveled the consecutive expression of antigens
(2–5,20). Refinements in multiparameter flow cytometry
allowed the analysis of large numbers of B cells by which
minor B-cell populations could be identified (4,5,34). The
two selected quadruple immunostainings have proved ex-
tremely useful for fast screening of the BM B-cell compart-
ment, enabling the identification of (pathological) abnor-
malities in B-cell differentiation.
Immunostaining 1: TdT/CD20/CD19/CD10. The
combination of CD10 and CD20 with a pan-B-cell marker
(CD19) is a well-established combination for analysis of
B-cell differentiation (2,4). Within the CD19⫹ B cells, at
least four sequential maturation stages can be discrimi-
nated based on CD10 and CD20 expression (Fig. 1A:
stages I–IV). These four differentiation stages form a con-
tinuous staining pattern in a CD10/CD20 plot (Fig. 1D) as
a result of the stepwise loss of CD10 and the gradual gain
of CD20 during maturation. Plasma cells, when present,
reveal a minor discrepant population in this dot plot,
being CD10⫺CD20⫺. As both CD10 and CD20 show a
wide range in expression levels during B-cell differentia-
tion, APC- or PE-conjugates of these mAb are preferred for
discrimination of the various subpopulations. Adding TdT
to the CD10/CD20 combination of mAb enables a better
discrimination between the immature CD10bright cells that
are TdT⫹ and the more mature CD10⫹ cells that are
TdT⫺(Fig. 1C). As detection of TdT concerns an intracel-
lular staining, the addition of TdT delays the otherwise
very quick screening. In our opinion, however, the advan-
tage of a better discrimination between the CD10⫹ pre-
cursor B-cell populations compensates the more complex
intracellular staining. First, a low expression of TdT com-
bined with a high CD10 expression is characteristic for
precursor B-ALL (20). Second, the ratio between
TdT⫹CD10⫹ and TdT⫺CD10⫹ precursor B cells can be
indicative for (drug-induced) regeneration of BM (9,10).
Immunostaining 2: CD45/CD34/CD19/CD22. The
second quadruple staining concerns the combination of
CD34 with CD22, and CD45 and CD19. Again, four major
B-cell subpopulations (Fig. 1A: stages I–IV) can be discrim-
inated within the CD19⫹ B cells. The most immature B
cells from the first immunostaining (TdT⫹CD10⫹ cells)
globally correspond to the CD34⫹CD22⫹CD45dim cells in
this immunostaining (Fig. 1E,F). The CD45 expression
increases during maturation followed by an increased
CD22 expression (Fig. 1F). The mature CD10⫺CD20⫹ B
cells from the first immunostaining globally correspond to
the CD22brightCD45bright cells in the CD22/CD45 dot plot.
When present, CD22⫹CD19⫺CD34⫹ pro-B cells can be
identified as a minor population in addition to the earlier
described four stages of B-cell differentiation (5,25).
In a standardized setting, with a calibrated flow cytom-
eter and using the same mAb clones, the staining patterns
from both immunostainings as displayed in the respective
plots are quite stable. Shifts in the relative sizes of the
subpopulations or a different location in the plot may
therefore indicate (ab)normal variations or aberrancies in
the B-cell development.
4 VAN LOCHEM ET AL.
Normal variations and abnormalities in B-cell dif-
ferentiation. The expression profiles from the two se-
lected immunostainings are quite stable, but shifts in the
relative sizes of the subpopulations can be found depen-
dent on the age of the individual (4). In children the
CD10⫹ subpopulations predominate, while in adults the
CD10⫺CD20⫹ B cells are more frequent. In the elderly
(generally ⬎75 years), the mature B-cell population can
even occupy most of the BM B-cell compartment (Fig.
2A–C). Moreover, the CD10⫺CD20⫺ plasma cells repre-
sent a substantial subpopulation of the CD19⫹ B cells in
the BM of an older person (cyan colored). These age-
related shifts should be regarded as normal.
FIG. 1. Normal B-cell development
in bone marrow (BM). A: In this
scheme for normal B-cell develop-
ment in BM, differentiation stages
are depicted that can be recognized
with the two selected four-color im-
munostainings 1 and 2. The colors in
the differentiation scheme (A) glo-
bally correspond to the colors in the
underlying dot plots (B–F) and repre-
sent different B-cell subpopulations
in childhood BM (4 year old healthy
child). To identify B cells, a CD19
gate was used in both immunostain-
ings: TdT/CD20/CD19/CD10 (B-D)
and CD45/CD34/CD19/CD22 (E,F).
Within the CD19⫹ B cells, at least
four subpopulations can be identified
with both immunostainings.
Irrespective of age, shifts in the relative distribution of the
(precursor) B-cell subpopulations can also be found in regen-
erating BM (Fig. 3A–C). Precursor B-cell regeneration can be
observed after chemotherapy for acute leukemia or during
temporary therapy stops. Differences in the precursor B-cell
regeneration patterns are known to be related to the inten-
sity of the preceding treatment (9,10). Following a high-
intensity therapy block such as induction treatment of ALL
patients, the more immature TdT⫹CD10⫹ precursor B cells
dominate over the TdT⫺CD10⫹ precursor B cells during BM
regeneration, whereas the ratio between the TdT⫹CD10⫹
and TdT⫺CD10⫹ precursor B cells is inversed when the
preceding therapy block has been less intense (9).
 IMMUNOPHENOTYPIC REFERENCE PATTERNS OF NORMAL BONE MARROW 5

FIGURE 2

FIGURE 3

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
FIG. 2. Age-related shift in B-cell compartment of elderly individuals.
In bone marrow (BM) of elderly individuals, the relative size of the
(CD10⫹) precursor B-cell population is decreased when compared to BM
of young individuals (see Fig. 1). The CD19⫹ B-cell population in the
adult BM (A) is predominated by the more mature TdT⫺CD10⫺CD20⫹ B
cells (B,C). The cyan dots represent the plasma cell population
(CD19⫹CD10⫺CD20⫺TdT⫺), which can be a substantial subpopulation
in the BM of elder individuals. Note the change in using CD19-PE
instead of CD19-PerCP, and CD20-PerCP instead of CD20-PE.
FIG. 3. Regenerating precursor B cells in bone marrow (BM) after
cytotoxic treatment. In BM from a patient treated for precursor B-acute
lymphoblastic leukemia (BALL), massive regeneration of the precursor
B-cell compartment is seen as soon as the therapy is (temporarily)
stopped after induction treatment. Analysis of CD19⫹ B cells (A) shows
high numbers of (nonleukemic) CD10⫹ precursor B cells in the BM
sample, with relatively low numbers of more mature CD10⫺CD20⫹ B
cells (B,C).
FIG. 4. Alternative immunostainings for normal B-cell development in
bone marrow (BM). Alternative immunostainings that show the different
stages in B-cell development are CyIg␮/SmIgM/CD20/CD10 and SmIg␬/
SmIg␭/CD20/CD10. When gated on CD10⫹ and CD20⫹ small cells
(lymphocyte scatter) (A,B), the stepwise gain of CyIg␮ and gradual in-
crease in SmIgM expression is observed (C) using alternative staining
CyIg␮/SmIgM/CD20/CD10: CyIg␮⫺CD10⫹CD20⫺ precursor B cells dif-
ferentiate to mature SmIgM⫹CD10⫺CD20⫹ B lymphocytes. Using a
comparable gating strategy, the differentiation into either Ig␬- or Ig␭
FIGURE 4 positive B cells can be shown (D) using alternative staining SmIg␬/
SmIg␭/CD20/CD10.
6 VAN LOCHEM ET AL.
Besides the shifts in the relative sizes of the subpopu-
lations, shifts in expression levels of markers can be
observed. This should be considered with great suspi-
cion. Precursor B-ALL frequently display aberrant
marker expression by which the malignant population
will occupy a so-called empty space of the dot plot
template: the cells are located in a space where nor-
mally no cells are found (20). Examples include very
high expression of CD10 combined with low expres-
sion or negativity for TdT or low expression or nega-
tivity for CD45 combined with a relatively high CD22
expression (20,35,36).
Other possible quadruple immunostainings for
studying B-cell differentiation. The two selected qua-
druple immunostainings (i.e., TdT/CD20/CD19/CD10 and
CD45/CD34/CD19/CD22) are efficient for first screening of
the B-cell compartment in BM. They quickly reveal shifts or
abnormal patterns in the B-cell differentiation profile, which
then need to be further analyzed in detail. It is obvious that
these two immunostainings are not the only possibilities for
screening of the B-cell compartment in BM.
In combination with CD10, CD20, and TdT, one can
argue the use of CD22 instead of CD19, as a (minor)
subpopulation of precursor B cells (pro-B cells) is
known to be CD19 negative (5). On the other hand,
normal plasma cells are generally CD22 negative
(37,38). Since both CD19⫺ and CD22⫺ B cells concern
only minor subpopulations of B cells, both markers are
useful as pan-B-cell markers for the major B-cell sub-
populations. In the two B-cell immunostainings we pre-
fer to use CD19 as the pan-B-cell marker for gating the
B-cell lineage, because of its homogeneous expression.
The expression of CD22 is much more heterogeneous,
varying from dim to bright positive, as is illustrated in
Figure 1F. This heterogeneous expression and the ex-
pression of CD22 on basophils (39) makes CD22-based
B-cell gating more complicated. In some cases, rela-
tively high background staining of CD19 can be seen on
monocytes: this is caused by binding of the mAb to FcR.
This background staining generally does not affect B-
cell gating if a combination of CD19 positivity with low
SSC characteristics is used as a selection gate.
As indicated, the intracellular TdT staining may delay
the screening process. For reasons of efficiency, the im-
munostainings CD45/CD20/CD19/CD10 or CD34/CD20/
CD19/CD10 will be reasonable alternatives to discrimi-
nate the various immature B-cell subsets, taking into
account that the expression patterns of TdT, CD45 and
CD34 are different (Fig. 1).
CD10 and CD20 can also be combined with many
other differentiation-stage specific markers like cyto-
plasmic immunoglobulin ␮ chain (CyIg␮), surface mem-
brane (Sm)IgM or SmIg light chain. These immunostain-
ings will give extended information about the
maturational stage of the various subpopulations (Fig.
4), but will not identify additional major B-cell subpopu-
lations in BM.
Monocytic Cell Differentiation
Normal monocytic differentiation. Immunostaining
of cells of the monocytic lineage can be hampered by the
aspecific binding of mAb via FcR on monocytes or by
autofluorescence of the monocytes. For specific staining
of more mature monocytes, IgG2a mAb should be avoided
whenever possible because monocytes express high lev-
els of the IgG2a binding FcR’s (CD64) (24).
The most immature monocytic precursor cell (mono-
blast) can immunophenotypically not be discriminated
from the most immature granulocytic precursor cell (my-
eloblast) or from their common progenitor. Immunostain-
ing 3 was selected to identify the myelo/monoblasts. Two
additional differentiation stages can immunophenotypi-
cally be identified with an immunostaining specific for
monocytic differentiation (Fig. 5A; immunostaining 4).
Immunostaining 3. CD34/CD117/CD45/CD13.33.
Both CD33 and CD13 are specific markers for the granu-
locytic and monocytic lineage. As their expression level is
heterogeneous during differentiation, a mixture of CD13
and CD33 mAb was used in this third immunostaining to
optimize the staining of all cells that belong to the gran-
ulocytic and monocytic lineage.
For the identification of myelo/monoblasts, a combination
of CD45 expression and SSC pattern is useful in the gating
procedure (Fig. 5B). A dim expression of CD45 is seen on
generally all precursor cells in BM: myeloblasts, monoblast,
precursor B cells, and erythroblasts. CD34 is expressed on
precursors-cells of granulocytic, monocytic and B-cell lin-
eage, while CD13.33 and CD117 are expressed predomi-
nantly on precursor cells of the granulocytic/monocytic lin-
eage. CD117 is also expressed on the (CD34dimCD13.33⫹)
promyelocytes (Fig. 5C), on a minor subpopulation of pre-
cursor B cells and at very high levels on mast cells (40 – 42).
The latter populations is however hardly detectable in nor-
mal BM. The CD34⫹CD117⫹CD45dimCD13.33⫹ cells in this
immunostaining represent the myelo/monoblasts, while the
majority of CD34⫹CD117⫺CD45dimCD13.33⫺ cells are pre-
cursor B cells (Fig. 5B,D).
Immunostaining 4. CD14/CD33/CD45/CD34. The
progression from monoblast to promonocyte is marked by
an increase in CD33 expression and the disappearing of
CD34 expression, while the cells maintain intermediate lev-
els of CD45 (Fig. 5E,G). The CD34⫺CD33highCD45intermediate
promonocytes express low levels of CD15 on their surface.
Subsequently, the CD45 expression is increased and the cells
gain CD14 (Fig. 5F). This CD14 expression marks the mono-
cyte stage. In contrast to the neutrophils, the monocytic cells
retain HLA-DR during their maturation.
Normal variations and abnormalities in mono-
cytic development. Shifts in the relative distribution of
the different immunophenotypically identified monocytic
subpopulations are rarely seen as a result of normal (age-
related) variations. Monoblasts or promonocytes are over-
represented in cases of monoblast-predominant AML
(AML-M5a) or promonocyte-predominant AML (AML-
M5b), respectively (Fig. 6). Consequently, the shifts in the
immunophenotypic profiles will be as prominent as the
 IMMUNOPHENOTYPIC REFERENCE PATTERNS OF NORMAL BONE MARROW 7

FIG. 5. Normal monocytic develop-

ment in bone marrow (BM). A: In this
scheme for normal monocytic devel-
opment in BM, differentiation stages
are depicted that can be identified by
the two selected four-color immuno-
stainings 3 and 4. The colors in the
differentiation scheme globally corre-
spond to the colors in the underlying
dot plots and represent different sub-
populations. B–D: Expression profiles
of immunostaining 3 (CD34/CD117/
CD45/CD13.33). A combination of
low CD45 expression and low SSC is
used to identify the precursor cells in
BM with immunostaining 3. The
CD34 ⫹ CD117 ⫹ CD45 dim CD13.33 ⫹
cells in this immunostaining repre-
sent the myelo/monoblasts (red
dots), the CD34⫹CD117⫺CD45dim
cells (blue dots) represent the pre-
cursor B cells. The green dots repre-
sent the promyelocytes (see also Fig.
7A), which can be identified with this
immunostaining by the increased
SSC and high CD13.33 expression
(D). Note that a mix of APC-conju-
gated CD13 and CD33 mAb is used
to optimize the staining of all cells
that belong to the granulocytic and
monocytic lineage. Normal mono-
cytic development in BM is illus-
trated with immunostaining 4 (CD14/
CD33/CD45/CD34), when an SSClow
gate is combined with a CD45low– high
gate (E). Three differentiation stages
can be discriminated including the
myelo/monoblast stage: CD34 ⫹
CD33 ⫹ CD14⫺ myelo/monoblasts,
CD34⫹/⫺CD33highCD14⫺ pro-mono-
cytes and CD34 ⫺ CD33 high CD14 ⫹
monocytes (F and G). LWBM: ammo-
niumchloride-lysed whole BM.

shifts in the relative frequency of the leukemic cell pop-
ulation. Maturation asynchronisms such as coexpression
of CD34/CD11b or CD34/CD15; cross-lineage marker ex-
pression such as CD19, CD2, or CD7; and aberrant inten-
sities of expression may be correlated with the prognosis
of the disease in AML (16,17,43).
In cases of myeloid dysplastic syndrome (MDS) shifts in
the frequency of monocytes (in normal BM, 1– 8%) can be
seen. Either monocytosis or a lack of monocytes can
distinguish different types of MDS (44,45). For those
cases, immunostaining 4 (CD14/CD33/CD45/CD34) is
useful to quantify the CD14⫹ CD45bright monocytes.
Other possible quadruple immunostainings for
studying monocytic differentiation. For evaluation of
monocytic development, CD36, CD64, or CD4 (dimly
expressed on monocytes) can be used in addition to
CD14. CD36 expression and dim CD4 expression often
precede CD14 expression and may indicate the mono-
cytic differentiation of an otherwise CD34⫹ AML. In addi-
tion, CD68 is useful to identify the final stage of monocytic
differentiation, i.e., macrophages. With their increased
FSC and SSC, macrophages can be distinguished from
their precursors. However, in BM generally no macro-
phages are detectable.
As mentioned before, transient dim CD15 expression is
seen on cells of the monocytic lineage preceding the
CD14 expression and is only detectable with high affinity
CD15 mAb (22,24). However, low expression of CD15 on
CD34⫹ precursor cells identifies not only commitment to
monocytic development, but also granulocytic precursor
cells in transition from CD34⫹CD15⫺ blasts to CD34⫺/
CD15bright promyelocytes.(46).
8 VAN LOCHEM ET AL.

FIG. 6. Acute myeloid leukemia

(AML) with monocytic differentia-
tion. bone marrow (BM) of a patient
with an acute monocytic leukemia.
Most cells in the BM are of monocytic
lineage. Monoblasts, promonocytes,
and monocytes can be recognized;
the promonocytes are most predomi-
nant. The dot plots illustrate the im-
munophenotypic heterogeneity within
an acute leukemia.

Granulocytic Cell Differentiation comprise only minor subpopulations in normal BM. As

Normal granulocytic (neutrophil) differentiation. mentioned before, the most immature granulocytic pre-
The granulocytic differentiation in BM generally concerns cursor cell (myeloblast) cannot be discriminated immuno-
neutrophil differentiation, as eosinophils and basophils phenotypically from the most immature monocytic pre-

FIG. 7. Normal granulocytic devel-

opment in bone marrow (BM).
A: Scheme for normal granulocytic
development in BM, depicting differ-
entiation stages that can be identi-
fied by the two selected four-color
immunostainings 3 and 5. The colors
in the differentiation scheme globally
correspond to the colors in the under-
lying dot plots and represent differ-
ent subpopulations. The expression
profiles of immunostaining 3 (CD34/
CD117/CD45/CD13.33) are pre-
sented in Fig. 5B–D. Light scatter
characteristics in combination with
the intermediate CD45 expression is
used to identify the granulocytic lin-
eage (B), in immunostaining 5
(CD16/CD13/CD45/CD11b). Five
differentiation stages are distinguish-
able, based on the gradual increase
of CD11b (E,F) and CD16 expression
(C,E,G) and the dynamic CD13 ex-
pression (F,G) in combination with
SSC characteristics (D). CD16 ex-
pression is preceded by the CD11b
expression (E).
 IMMUNOPHENOTYPIC REFERENCE PATTERNS OF NORMAL BONE MARROW
cursor cell (monoblast). One of the two immunostainings
we selected for the monocytic differentiation (immuno-
staining 3) identifies both the monocytic and granulocytic
precursor cells, while the other (immunostaining 5) un-
ravels the further granulocytic differentiation.
Immunostaining 3: CD34/CD117/CD45/CD13.33.
For screening of abnormalities in the immature granulo-
cytic/monocytic precursor cells, we selected a quadruple
staining that covers the early precursors of both lineages:
combination of the precursor markers CD34 and CD117
with CD45 and a mixture of CD13 and CD33 will reveal
eventual shifts in the relative size or immunophenotype of
the myelo/monoblasts (Fig. 5B–D). This immunostaining
also identifies promyelocytes as CD117⫹CD34dim
CD13.33high cells with an increased SSC.
Immunostaining 5: CD16/CD13/CD45/CD11b. The
second quadruple immunostaining for the granulocytic
differentiation concerns the combination of CD45 and
CD13 with CD16 and CD11b. An intermediate CD45 ex-
pression discriminates the cells of the granulocytic lineage
from the CD45high lymphocytes and monocytes. CD45
expression, in combination with the granulocytic/mono-
cytic lineage marker CD13 and light scatter characteris-
tics, is useful to select and study the granulocytic differ-
entiation patterns (45,47).
CD13 is a unique marker in the granulocytic differenti-
ation as it is dynamically expressed during granulocytic
differentiation. It is expressed at high levels on myelo-
blasts and promyelocytes. CD13 is downregulated and
dimly expressed on myelocytes and is gradually upregu-
lated again as the granulocytic cells develop into seg-
mented neutrophils (Fig. 7D). Combination of this dy-
namic marker with CD11b and CD16 in one
immunostaining reveals four sequential maturation stages
in the neutrophil development (Fig. 7A: stage II–V; Fig.
7E–G). CD11b and CD16 are initially expressed at low
levels, but their expression increases during the develop-
ment process, particularly in the last 2 stages (stages IV
and V; Fig. 7E).
With the quadruple immunostaining 5 (CD16/CD13/
CD45/CD11b), the myelo/monoblasts can be identified as
CD16⫺CD13⫹CD45intermediateCD11b⫺. They have an inter-
mediate forward scatter (FSC) but still a low SSC and
globally correspond to the CD34⫹CD117⫹CD45intermediate
CD13.33⫹ cells from immunostaining 3 (Fig. 7). When the
myeloblasts progress to the stage of promyelocytes, a
dramatic increase in SSC is seen (Fig. 7B,D). From this
moment, the cells express CD15 and retain their high SSC.
In the subsequent maturation stages CD11b is acquired,
followed by CD16 (Fig. 7E–G). Note that the relatively
high fluorescence signal for CD16-fluoroscein isothiocya-
nate (FITC) of promyelocytes and myelocytes in Figure
7E,G has nothing to do with early CD16 expression but
with the high autofluorescence of these subpopulations in
the FL1 (FITC) channel.
Like B-cell development, the staining patterns of immu-
nostaining 5 for normal granulocytic differentiation in BM,
are quite stable with regard to the immunophenotypic
characteristics and relative distribution of the various sub-
9
populations (provided that the same mAb clones are
used).
Normal variations and abnormalities in neutro-
phil differentiation. The myeloid differentiation mainly
concerns the maturation to neutrophilic granulocytes, as
the other end-stage granulocytes (i.e., the basophils and
eosinophils) comprise only minor cell populations in nor-
mal BM. However, as a result of immune responses to
specific agents or in case of a chronic myeloid leukemia
(CML), shifts can be observed in the relative distribution
of the various end-stage granulocytes (48,49). In BM of a
patient with a CML-accelerated phase, eosinophils and
basophils can be identified as prominent subpopulations
of the granulocytic lineage (Fig. 8). On the FSC/SSC dot
plot, eosinophils show a high SSC and lower FSC com-
pared to the neutrophils (Fig. 8A). Expression of CD16 on
eosinophils is dim and also expression of CD11b and
CD13 is somewhat lower compared to neutrophils (Fig.
8C). In contrast, CD45 expression on eosinophils is con-
sistently higher then on neutrophils (Fig. 8B). Basophils
have a much lower SSC compared to the other granulo-
cytic subpopulations and are hard to distinguish from
lymphocytes and, to a lesser extend, monocytes based on
light scatter characteristics. An intermediate CD13 expres-
sion on basophils (high on monocytes and absent on
lymphocytes) and a lower CD45 expression (Fig. 8B), help
identify these cells. Furthermore, basophils have a unique
immunophenotype, different from the other granulocytic
cells. They express CD22, but are negative for other B-cell
markers (39). An increase in the relative size of the baso-
philic cell population can be seen in the chronic and
accelerated phase of CML (48).
Shifts in the relative distribution of the granulocytic
subpopulations and in the immunophenotypic character-
istics are seen in myeloid malignancies like AML and
myeloid dysplastic syndrome (MDS) (50,51). An increase
of immature granulocytic precursor cells or a reduction in
well-differentiated granulocytes can be seen in both dis-
eases. The two immunostainings were not selected for the
identification of all sorts of immunophenotypic aberran-
cies that can be expected in patients with AML or MDS,
but they are useful for a quick screening of BM for such
abnormalities. First, immunostaining 3 (CD34/CD117/
CD45/CD13.33) will illustrate that most CD34⫹ precursor
cells in MDS or AML BM belong to the myelo/monocytic
lineage, and not to the B-cell lineage, as they are CD117⫹
and/or CD13.33⫹. In addition, the intermediate CD45
expression in combination with SSC identifies increased
numbers of myelo/monoblast cells in both immunostain-
ings. Second, morphological abnormalities like hypo- or
hypergranulation will be reflected in reduced or increased
SSC values (Fig. 9). Third, in MDS an overexpression of
CD13 and CD33 and a reduced expression of CD11b and
CD16 is frequently seen and consequently the CD13/
CD16 and CD13/CD11b dot plots of immunostaining 5
will show an aberrant expression profile of the granulo-
cytic cells (Fig. 9).
10 VAN LOCHEM ET AL.

FIG. 8. Eosinophilic and basophilic granulocytes in bone marrow (BM) of a chronic myeloid leukemia (CML) patient. In BM of a patient with
CML-accelerated phase, relatively high numbers of eosinophilic (yellow dots) and basophilic (red dots) granulocytes can be identified. Eosinophilic
granulocytes occupy a discrepant position in the forward scatter/side scatter (FSC/SSC) dot plot (A) and show a CD45 expression comparable to
monocytes (B; blue dots). Basophilic granulocytes are hard to distinguish from lymphocytes in a FSC/SSC dot plot (A; green dots). An intermediate CD45
expression comparable to neutrophilic granulocytes however, discriminates the basophilic granulocytes (B). In the CD11b/CD13 dot plot, the basophilic
and eosinophilic granulocytes show a slightly lower CD11b expression than the mature neutrophilic granulocytes (c).

Other possible quadruple immunostainings for
studying granulocytic differentiation. As mentioned
before, both CD33 and CD13 are specific markers for the
granulocytic and monocytic lineage that can be expressed
heterogeneously during differentiation. To optimize the
staining of all cells that belong to the granulocytic and
monocytic lineage, a mixture of CD13 and CD33 mAb can
be used. An aberrant cross-lineage expression of CD13
and/or CD33 is seen in 40 – 60% of precursor B-ALL pa-
tients. CD65 can be used as an alternative pan-myeloid
marker to cover a large part of the granulocytic differen-
tiation from myeloblasts, until end-stage granulocytes.
Although described as a pan-granulocytic marker, CD15
expression is not confined to the granulocytic differentia-
tion. In precursor cells committed to the monocytic differ-
entiation (like promonocytes), CD15 can be expressed at
low levels. Low-affinity mAb will only stain the granulocytic
lineage, while high-affinity mAb (e.g., clone MM1; BD Bio-
sciences) will also stain the immature monocytic cells. This
mAb is useful in combination with CD34 and monocytic
markers to identify the monocytic precursor cells.
CD66b expression precedes CD16 expression compa-
rable to CD11b, being present on (pro-) myelocytes. Con-
sequently, CD66b in combination with a pan-myeloid
marker, like CD13 or CD33, will also reveal different
stages in the granulocytic differentiation and is therefore
an alternative mAb for CD11b. We selected the combina-
tion of CD11b with CD16 as CD11b distinguishes the early
differentiation stages in granulocytic differentiation very
well, whereas CD16 distinguishes the more mature differ-
entiation stages very well. In addition to CD16 and
CD11b, other markers that are not restricted to the gran-
ulocytic or monocytic lineage, like CD64 or CD35, will
also show variations in their expression levels during
granulocytic differentiation and are useful in combination
with CD13 and/or CD33 (52).
Erythroid differentiation
Normal erythroid differentiation. To minimize
noise when measuring leukocytes, erythrocytes in BM sam-
ples are generally lysed. The many commercially available
permeabilizing or lysing reagents and ammonium chloride
(NH4Cl) preserve the lyse-resistant nucleated red cells of the
erythroid lineage for immunophenotypic analyses.

FIG. 9. Hypogranular granulocytes in bone marrow (BM) of a MDS patient. BM of an MDS patient displays various aberrancies in the granulocytic
lineage. Hypogranularity of the granulocytes results in a reduced SSC (A). Furthermore, the staining patterns in the CD11b/CD13 and CD13/CD16 dot
plots are clearly changed with strongly reduced promyelocytic and myelocytic subsets (B,C).
 IMMUNOPHENOTYPIC REFERENCE PATTERNS OF NORMAL BONE MARROW 11

FIG. 10. Normal erythroid development in bone mar-

row (BM). A: In this scheme for normal erythroid devel-
opment in BM, differentiation stages are depicted that
can be identified by the selected four-color immuno-
staining 6. The colors in the differentiation scheme
globally correspond to the colors in the underlying dot
plots and represent different subpopulations. When
gated on SSClow in combination with CD45neg-low, only
the erythroblasts are seen in normal BM (B,C). Pro-
erythroblasts will form a minor population in normal
BM and erythrocytes are lysed in the sample procedure.

Immunostaining 6. CD71/CD235a/CD45/CD117. Normal variations or abnormalities in erythroid

This selected quadruple immunostaining clearly identifies
two sequential differentiation stages of nonlysed nucle-
ated erythroid cells in BM samples after red cell lysis (Fig.
10A). Cells of the erythroid lineage are generally identified
by the gradual loss of CD45 (6). Only the CD117⫹ CD71⫹
erythroid-precursors and pro-erythroblasts do express
CD45 dimly. During maturation, the expression of the
transferrin receptor CD71 slightly increases, where-after
CD235a (glycophorin A) is being expressed. The red cells
retain CD235a but lose CD71 together with loss of the
nucleus (6,31).
development. The nucleated erythroid population can
be much more predominant in cases of a severe aplastic
anemia or MDS, or during BM regeneration after drug-
induced BM suppression. In regenerating BM of children
treated for ALL, the erythroid cell population can occupy
up to 50% of the cells in the lysed BM sample (53). These
CD45⫺CD71⫹CD235a⫹ cells include both the nucleated
erythroid cells and the reticulocytes, which tend to be
more resistant to lysis when present in high numbers in
young children (Fig. 11A–C). Although shifts in the relative
frequency of nucleated red cells can be seen in regenerating

FIG. 11. Erythroid development in bone marrow (BM) of a patient with regenerating BM. In regenerating BM of a patient treated for acute lymphoblastic
leukemia (ALL), three differentiation stages can be discriminated in the erythroid development: CD117⫹CD45lowCD71⫹CD235a⫺ pro-erythroblasts (red
dots), CD117⫺CD45neg-lowCD71⫹CD235a⫹ erythroblasts (green) and CD117⫺CD45⫺CD71⫺CD235a⫹ nonlysed erythrocytes (blue dots) can be
discriminated.
12 VAN LOCHEM ET AL.
BM, severe anemia, megaloblastic anemia or brisk hemolytic
anemia, or BM of very young children, immunophenotypic
shifts as a result of aberrant marker expression are rare. In
contrast, in MDS patients aberrant expression of CD71
and/or CD235a is frequently observed (50).
CONCLUDING REMARKS
We present six quadruple immunostainings that detect all
major leukocyte populations in BM. All six quadruple immu-
nostainings reveal remarkably stable immunostaining pat-
terns and are therefore useful as a frame of reference for
quick identification of shifts in normal BM development.
These shifts can either be age-related or disease- or drug-
induced. Age-related shifts in normal BM development gen-
erally concern shifts in the relative distribution of the various
subpopulations within the respective lineages. Although sev-
eral subpopulations may become more predominant than
others during aging, the shape of the immunophenotypic
staining pattern remains unaltered. Alterations in the relative
distribution of the subpopulations can also be seen in regen-
erating or reactive BM when erythroid, precursor B-cell or
myelo/monoblast populations are clearly overrepresented.
In most cases of malignant hematopoiesis, such as MDS or
acute leukemia, the aberrant immunophenotype often posi-
tions the malignant cell population at a separate place in the
dot plot. Such immunophenotypic shifts should therefore
always be considered as an abnormality in the hematopoie-
sis. Further detailed flow cytometric analyses of the relevant
cell lineage is then recommended.
In this report we do not include immunostainings for
identification of the NK-/T-cell lineage, since NK-/T-cell
differentiation does not take place in BM. The T-cell pop-
ulation found in normal BM comprises mature blood de-
rived T lymphocytes. T-cell immunostainings of normal
BM samples will therefore reveal nondynamic immuno-
staining patterns with a homogeneous T-cell population
and no clear subpopulations other than CD4⫹ and CD8⫹
T lymphocytes. Adding a single T-cell marker to the six
selected quadruple stainings will not give additional infor-
mation, as changes in the relative number of T cells in the
BM will mainly reflect the quality of the aspirated BM.
However, for screening BM involvement in immature or
mature T-cell malignancies, we suggest the addition of
two specific T-cell immunostainings: CD2/CD5/CD3/CD7
and CD4/CD8/CD3/HLA-DR (19).
Reference values for the major leukocyte subpopulations
in BM are only partly available, but the dot plot templates of
the six selected immunostainings can serve as a reference.
The normal immunophenotypic expression profiles help
identify normal and abnormal variations in hematopoiesis
and are therefore useful as first screening.
ACKNOWLEDGMENTS
The authors thank Marieke Comans-Bitter for preparing
the figures; the hematologists and pediatricians for sub-
mitting patient samples to our laboratory; and Ivonne
Bronmans, Anja de Jong, Jac Kuijpers-Entrup, Marja Smits,
Dennis Tielemans, and Patricia Hoogeveen for their excel-
lent technical support.
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