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Immunophenotypic differentiation patterns of

normal Hematopoiesis in human bone marrow:
Reference patterns for...

Article in Cytometry Part B Clinical Cytometry July 2004

DOI: 10.1002/cyto.b.20008 Source: PubMed


147 248

6 authors, including:

Ellen van Lochem Vincent H J van der Velden

Rijnstate Hospital Erasmus MC


Nomdo Westerdaal
Det Norske Radiumhospitalet


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Cytometry Part B (Clinical Cytometry) 60B:113 (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 ow cytometry allow stepwise analysis of bone marrow (BM) samples that are suspected for abnormal
hematopoiesis. A screening phase that precedes lineage-specic classication phases should be sufcient
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 ow 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 specic 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
identication of shifts in the composition of the studied differentiation lineages, reecting age-related
changes or disease-induced BM abnormalities. 2004 Wiley-Liss, Inc.

Key terms: ow cytometry; normal bone marrow; staining patterns; immunophenotyping; expression prole

Flow cytometric analysis of bone marrow (BM) samples mature markers in normal hematopoietic development
is performed routinely for identication, frequency assess- provides a frame of reference for recognition of abnormal
ment, and further characterization of the various leuko- differentiation patterns.
cyte subpopulations when abnormal hematopoiesis is sus- Abnormal BM hematopoiesis may result from (1) an
pected. The BM compartment is a complex tissue arrest during precursor B-cell differentiation; (2) BM re-
containing cells of multiple hematopoietic lineages with generation after immunosuppressive therapy or as a re-
various maturational stages per lineage. In healthy individ- sponse to infection; (3) dysplasia in one or more myeloid
uals, the BM precursor cells guarantee continuous produc- lineages; (4) increased turnover of hematopoietic cells
tion of the various hematopoietic differentiation lineages.
The differentiation and maturation can be monitored by
changes in cytomorphology and immunophenotype. Es- *Correspondence to: E.G. van Lochem, Department of Immunology,
pecially in the B-cell lineage, multiple stages have been Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein
dened based on their immunophenotype (15). Also in 40, 3015 DR Rotterdam, The Netherlands.
the monocytic, granulocytic, erythroid and thrombocytic E-mail:
Received 18 August 2003; Accepted 29 October 2003
lineages several differentiation stages can be identied by Published online 14 June 2004 in Wiley InterScience (www.
immunophenotyping (6 8). Knowledge of the expres-
sion (levels) of various lineage-specic, immature, and DOI: 10.1002/cyto.b.20008

2004 Wiley-Liss, Inc.


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 specic 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 (1116). 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 (1720). 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 ow cytometry to the routine CD71 LO1.1 FITC BD Biosciences
use of four-color analyses. To generate signicant 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- specic immunoglobulin subclasses or by using F(ab)2
ture differentiation stages; a specic lineage (B- or myeloid fragments, thereby avoiding interaction with FcRs (24).
cells); or, a specic differentiation stage within a lineage. Third, most mAb are available with different uoro-
Numerous leukocyte antigens have been studied exten- chrome-conjugates; the choice of uorochrome-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 uorescence signal, PE- and APC-
aim to review the complete expression proles 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 uorochrome-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 identication 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 uorochromes. 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
afnity (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-specic immunostainings were included.
variable sensitivity for enzymes (23). Second, aspecic In ve of the six selected immunostainings, a lineage-
Fc-receptor (FcR) binding, causing an increased back- specic 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)
characteristics in a dot plot is useful to select and study further unraveled the consecutive expression of antigens
the immunophenotypic differentiation patterns within the (25,20). Renements in multiparameter ow cytometry
B cell and granulocytic/monocytic lineage, respectively. allowed the analysis of large numbers of B cells by which
Apart from lineage-specic markers and immature or ma- minor B-cell populations could be identied (4,5,34). The
ture markers that are restricted to a particular cell lineage, two selected quadruple immunostainings have proved ex-
markers with a much broader expression pattern can be tremely useful for fast screening of the BM B-cell compart-
useful in the study of normal differentiation patterns in ment, enabling the identication of (pathological) abnor-
BM. The pan-leukocyte marker CD45 gives additional in- malities in B-cell differentiation.
formation about the major subpopulations, as the CD45 Immunostaining 1: TdT/CD20/CD19/CD10. The
antigen is differently expressed in various lineages and combination of CD10 and CD20 with a pan-B-cell marker
differentiation stages (6,27,28). CD45 expression is high (CD19) is a well-established combination for analysis of
on lymphocytes and monocytes, whereas granulocytes, B-cell differentiation (2,4). Within the CD19 B cells, at
precursor B cells, precursor granulocytic cells, and pro- least four sequential maturation stages can be discrimi-
erythroblasts are also CD45 positive, but at lower levels nated based on CD10 and CD20 expression (Fig. 1A:
(29,30). In contrast, (more) mature erythroid cells are stages IIV). These four differentiation stages form a con-
generally CD45 negative (6,31). Therefore, when com- tinuous staining pattern in a CD10/CD20 plot (Fig. 1D) as
bined with SSC characteristics, CD45 can help distinguish a result of the stepwise loss of CD10 and the gradual gain
the major leukocyte (sub)populations in BM. CD34 is of CD20 during maturation. Plasma cells, when present,
another non-lineage-restricted marker, expressed by the reveal a minor discrepant population in this dot plot,
precursor stages of the various cell lineages in BM (32,33). being CD10CD20. As both CD10 and CD20 show a
The normal differentiation patterns within a specic lin- wide range in expression levels during B-cell differentia-
eage can be displayed by the application of dynamic tion, APC- or PE-conjugates of these mAb are preferred for
markers, i.e., markers that are gradually up- or downregu- discrimination of the various subpopulations. Adding TdT
lated during differentiation. The immunophenotypic to the CD10/CD20 combination of mAb enables a better
changes are best illustrated when mAb directed against discrimination between the immature CD10bright cells that
markers with overlapping expression proles are com-
are TdT and the more mature CD10 cells that are
bined in one staining. For example, CD10 and CD20 (in
TdT(Fig. 1C). As detection of TdT concerns an intracel-
B-cell development) and CD13 and CD16 (in myeloid
lular staining, the addition of TdT delays the otherwise
development) are informative combinations (4,7). Ideally,
very quick screening. In our opinion, however, the advan-
the selected antibody combinations cover multiple con-
tage of a better discrimination between the CD10 pre-
secutive differentiation stages, resulting in dot plots that
cursor B-cell populations compensates the more complex
provide direct insight in the completeness and relative
intracellular staining. First, a low expression of TdT com-
distribution of the major subpopulations in the differenti-
bined with a high CD10 expression is characteristic for
ation lineage under study.
The composition of the selected immunostainings was precursor B-ALL (20). Second, the ratio between
based on our extensive experience obtained during the TdTCD10 and TdTCD10 precursor B cells can be
last decade with 900 1,200 BM samples analysed each indicative for (drug-induced) regeneration of BM (9,10).
year. All ow cytometric analyses of BM samples were Immunostaining 2: CD45/CD34/CD19/CD22. The
performed routinely with three-color immunostainings second quadruple staining concerns the combination of
during 1996 1999 and with four-color immunostainings CD34 with CD22, and CD45 and CD19. Again, four major
from 1999 until the present. B-cell subpopulations (Fig. 1A: stages IIV) can be discrim-
We selected a set of six four-color immunostainings for inated within the CD19 B cells. The most immature B
the analysis of the B-cell, monocytic, granulocytic and cells from the rst immunostaining (TdTCD10 cells)
erythroid cell lineages. Per lineage, the major subpopula- globally correspond to the CD34CD22CD45dim cells in
tions are described and illustrated in the respective plots this immunostaining (Fig. 1E,F). The CD45 expression
and differentiation schemes. Normal age-related variations increases during maturation followed by an increased
in the expression proles are discussed followed by ex- CD22 expression (Fig. 1F). The mature CD10CD20 B
amples of disease-induced shifts. We also discuss some cells from the rst immunostaining globally correspond to
alternative markers or four-color immunostainings that the CD22brightCD45bright cells in the CD22/CD45 dot plot.
can be used for the same purpose. When present, CD22CD19CD34 pro-B cells can be
identied as a minor population in addition to the earlier
BONE MARROW DIFFERENTIATION PATTERNS described four stages of B-cell differentiation (5,25).
B-Cell Differentiation In a standardized setting, with a calibrated ow cytom-
Normal B-cell differentiation. Early B-cell differenti- eter and using the same mAb clones, the staining patterns
ation has been extensively studied in BM using ow cyto- from both immunostainings as displayed in the respective
metric immunophenotyping. Initially, most studies fo- plots are quite stable. Shifts in the relative sizes of the
cused on precursor B-acute lymphoblastic leukemia subpopulations or a different location in the plot may
(precursor B-ALL) to unravel early B-cell development. therefore indicate (ab)normal variations or aberrancies in
Later, studies on the B-cell compartment in normal BM the B-cell development.

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 (BF) 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 identied
with both immunostainings.

Normal variations and abnormalities in B-cell dif- Irrespective of age, shifts in the relative distribution of the
ferentiation. The expression proles from the two se- (precursor) B-cell subpopulations can also be found in regen-
lected immunostainings are quite stable, but shifts in the erating BM (Fig. 3AC). Precursor B-cell regeneration can be
relative sizes of the subpopulations can be found depen- observed after chemotherapy for acute leukemia or during
dent on the age of the individual (4). In children the temporary therapy stops. Differences in the precursor B-cell
CD10 subpopulations predominate, while in adults the regeneration patterns are known to be related to the inten-
CD10CD20 B cells are more frequent. In the elderly sity of the preceding treatment (9,10). Following a high-
(generally 75 years), the mature B-cell population can intensity therapy block such as induction treatment of ALL
even occupy most of the BM B-cell compartment (Fig. patients, the more immature TdTCD10 precursor B cells
2AC). Moreover, the CD10CD20 plasma cells repre- dominate over the TdTCD10 precursor B cells during BM
sent a substantial subpopulation of the CD19 B cells in regeneration, whereas the ratio between the TdTCD10
the BM of an older person (cyan colored). These age- and TdTCD10 precursor B cells is inversed when the
related shifts should be regarded as normal. preceding therapy block has been less intense (9).



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 TdTCD10CD20 B
cells (B,C). The cyan dots represent the plasma cell population
(CD19CD10CD20TdT), 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 CD10CD20 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: CyIgCD10CD20 precursor B cells dif-
ferentiate to mature SmIgMCD10CD20 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/

Besides the shifts in the relative sizes of the subpopu- Monocytic Cell Differentiation
lations, shifts in expression levels of markers can be Normal monocytic differentiation. Immunostaining
observed. This should be considered with great suspi- of cells of the monocytic lineage can be hampered by the
cion. Precursor B-ALL frequently display aberrant aspecic binding of mAb via FcR on monocytes or by
marker expression by which the malignant population autouorescence of the monocytes. For specic staining
will occupy a so-called empty space of the dot plot of more mature monocytes, IgG2a mAb should be avoided
template: the cells are located in a space where nor- whenever possible because monocytes express high lev-
mally no cells are found (20). Examples include very els of the IgG2a binding FcRs (CD64) (24).
high expression of CD10 combined with low expres- The most immature monocytic precursor cell (mono-
sion or negativity for TdT or low expression or nega- blast) can immunophenotypically not be discriminated
tivity for CD45 combined with a relatively high CD22 from the most immature granulocytic precursor cell (my-
expression (20,35,36). eloblast) or from their common progenitor. Immunostain-
Other possible quadruple immunostainings for ing 3 was selected to identify the myelo/monoblasts. Two
studying B-cell differentiation. The two selected qua- additional differentiation stages can immunophenotypi-
druple immunostainings (i.e., TdT/CD20/CD19/CD10 and cally be identied with an immunostaining specic for
CD45/CD34/CD19/CD22) are efcient for rst screening of monocytic differentiation (Fig. 5A; immunostaining 4).
the B-cell compartment in BM. They quickly reveal shifts or Immunostaining 3. CD34/CD117/CD45/CD13.33.
abnormal patterns in the B-cell differentiation prole, which Both CD33 and CD13 are specic markers for the granu-
then need to be further analyzed in detail. It is obvious that locytic and monocytic lineage. As their expression level is
these two immunostainings are not the only possibilities for heterogeneous during differentiation, a mixture of CD13
screening of the B-cell compartment in BM. and CD33 mAb was used in this third immunostaining to
In combination with CD10, CD20, and TdT, one can optimize the staining of all cells that belong to the gran-
argue the use of CD22 instead of CD19, as a (minor) ulocytic and monocytic lineage.
subpopulation of precursor B cells (pro-B cells) is For the identication of myelo/monoblasts, a combination
known to be CD19 negative (5). On the other hand, of CD45 expression and SSC pattern is useful in the gating
normal plasma cells are generally CD22 negative procedure (Fig. 5B). A dim expression of CD45 is seen on
(37,38). Since both CD19 and CD22 B cells concern generally all precursor cells in BM: myeloblasts, monoblast,
only minor subpopulations of B cells, both markers are precursor B cells, and erythroblasts. CD34 is expressed on
useful as pan-B-cell markers for the major B-cell sub- precursors-cells of granulocytic, monocytic and B-cell lin-
populations. In the two B-cell immunostainings we pre- eage, while CD13.33 and CD117 are expressed predomi-
fer to use CD19 as the pan-B-cell marker for gating the nantly on precursor cells of the granulocytic/monocytic lin-
B-cell lineage, because of its homogeneous expression. eage. CD117 is also expressed on the (CD34dimCD13.33)
The expression of CD22 is much more heterogeneous, promyelocytes (Fig. 5C), on a minor subpopulation of pre-
cursor B cells and at very high levels on mast cells (40 42).
varying from dim to bright positive, as is illustrated in
The latter populations is however hardly detectable in nor-
Figure 1F. This heterogeneous expression and the ex-
mal BM. The CD34CD117CD45dimCD13.33 cells in this
pression of CD22 on basophils (39) makes CD22-based
immunostaining represent the myelo/monoblasts, while the
B-cell gating more complicated. In some cases, rela-
majority of CD34CD117CD45dimCD13.33 cells are pre-
tively high background staining of CD19 can be seen on
cursor B cells (Fig. 5B,D).
monocytes: this is caused by binding of the mAb to FcR.
Immunostaining 4. CD14/CD33/CD45/CD34. The
This background staining generally does not affect B- progression from monoblast to promonocyte is marked by
cell gating if a combination of CD19 positivity with low an increase in CD33 expression and the disappearing of
SSC characteristics is used as a selection gate. CD34 expression, while the cells maintain intermediate lev-
As indicated, the intracellular TdT staining may delay els of CD45 (Fig. 5E,G). The CD34CD33highCD45intermediate
the screening process. For reasons of efciency, the im- promonocytes express low levels of CD15 on their surface.
munostainings CD45/CD20/CD19/CD10 or CD34/CD20/ Subsequently, the CD45 expression is increased and the cells
CD19/CD10 will be reasonable alternatives to discrimi- gain CD14 (Fig. 5F). This CD14 expression marks the mono-
nate the various immature B-cell subsets, taking into cyte stage. In contrast to the neutrophils, the monocytic cells
account that the expression patterns of TdT, CD45 and retain HLA-DR during their maturation.
CD34 are different (Fig. 1). Normal variations and abnormalities in mono-
CD10 and CD20 can also be combined with many cytic development. Shifts in the relative distribution of
other differentiation-stage specic markers like cyto- the different immunophenotypically identied monocytic
plasmic immunoglobulin chain (CyIg), surface mem- subpopulations are rarely seen as a result of normal (age-
brane (Sm)IgM or SmIg light chain. These immunostain- related) variations. Monoblasts or promonocytes are over-
ings will give extended information about the represented in cases of monoblast-predominant AML
maturational stage of the various subpopulations (Fig. (AML-M5a) or promonocyte-predominant AML (AML-
4), but will not identify additional major B-cell subpopu- M5b), respectively (Fig. 6). Consequently, the shifts in the
lations in BM. immunophenotypic proles will be as prominent as the

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 identied 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. BD: Expression proles
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 CD34CD117CD45dim
cells (blue dots) represent the pre-
cursor B cells. The green dots repre-
sent the promyelocytes (see also Fig.
7A), which can be identied 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- CD14. CD36 expression and dim CD4 expression often
ulation. Maturation asynchronisms such as coexpression precede CD14 expression and may indicate the mono-
of CD34/CD11b or CD34/CD15; cross-lineage marker ex- cytic differentiation of an otherwise CD34 AML. In addi-
pression such as CD19, CD2, or CD7; and aberrant inten- tion, CD68 is useful to identify the nal stage of monocytic
sities of expression may be correlated with the prognosis differentiation, i.e., macrophages. With their increased
of the disease in AML (16,17,43). FSC and SSC, macrophages can be distinguished from
In cases of myeloid dysplastic syndrome (MDS) shifts in their precursors. However, in BM generally no macro-
the frequency of monocytes (in normal BM, 1 8%) can be phages are detectable.
seen. Either monocytosis or a lack of monocytes can As mentioned before, transient dim CD15 expression is
distinguish different types of MDS (44,45). For those seen on cells of the monocytic lineage preceding the
cases, immunostaining 4 (CD14/CD33/CD45/CD34) is CD14 expression and is only detectable with high afnity
useful to quantify the CD14 CD45bright monocytes. CD15 mAb (22,24). However, low expression of CD15 on
Other possible quadruple immunostainings for CD34 precursor cells identies not only commitment to
studying monocytic differentiation. For evaluation of monocytic development, but also granulocytic precursor
monocytic development, CD36, CD64, or CD4 (dimly cells in transition from CD34CD15 blasts to CD34/
expressed on monocytes) can be used in addition to CD15bright promyelocytes.(46).

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-
ed 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
proles of immunostaining 3 (CD34/
CD117/CD45/CD13.33) are pre-
sented in Fig. 5BD. 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).
cursor cell (monoblast). One of the two immunostainings populations (provided that the same mAb clones are
we selected for the monocytic differentiation (immuno- used).
staining 3) identies both the monocytic and granulocytic Normal variations and abnormalities in neutro-
precursor cells, while the other (immunostaining 5) un- phil differentiation. The myeloid differentiation mainly
ravels the further granulocytic differentiation. concerns the maturation to neutrophilic granulocytes, as
Immunostaining 3: CD34/CD117/CD45/CD13.33. the other end-stage granulocytes (i.e., the basophils and
For screening of abnormalities in the immature granulo- eosinophils) comprise only minor cell populations in nor-
cytic/monocytic precursor cells, we selected a quadruple mal BM. However, as a result of immune responses to
staining that covers the early precursors of both lineages: specic agents or in case of a chronic myeloid leukemia
combination of the precursor markers CD34 and CD117 (CML), shifts can be observed in the relative distribution
with CD45 and a mixture of CD13 and CD33 will reveal of the various end-stage granulocytes (48,49). In BM of a
eventual shifts in the relative size or immunophenotype of patient with a CML-accelerated phase, eosinophils and
the myelo/monoblasts (Fig. 5BD). This immunostaining
basophils can be identied as prominent subpopulations
also identies promyelocytes as CD117CD34dim
of the granulocytic lineage (Fig. 8). On the FSC/SSC dot
CD13.33high cells with an increased SSC.
plot, eosinophils show a high SSC and lower FSC com-
Immunostaining 5: CD16/CD13/CD45/CD11b. The
pared to the neutrophils (Fig. 8A). Expression of CD16 on
second quadruple immunostaining for the granulocytic
differentiation concerns the combination of CD45 and eosinophils is dim and also expression of CD11b and
CD13 with CD16 and CD11b. An intermediate CD45 ex- CD13 is somewhat lower compared to neutrophils (Fig.
pression discriminates the cells of the granulocytic lineage 8C). In contrast, CD45 expression on eosinophils is con-
from the CD45high lymphocytes and monocytes. CD45 sistently higher then on neutrophils (Fig. 8B). Basophils
expression, in combination with the granulocytic/mono- have a much lower SSC compared to the other granulo-
cytic lineage marker CD13 and light scatter characteris- cytic subpopulations and are hard to distinguish from
tics, is useful to select and study the granulocytic differ- lymphocytes and, to a lesser extend, monocytes based on
entiation patterns (45,47). light scatter characteristics. An intermediate CD13 expres-
CD13 is a unique marker in the granulocytic differenti- sion on basophils (high on monocytes and absent on
ation as it is dynamically expressed during granulocytic lymphocytes) and a lower CD45 expression (Fig. 8B), help
differentiation. It is expressed at high levels on myelo- identify these cells. Furthermore, basophils have a unique
blasts and promyelocytes. CD13 is downregulated and immunophenotype, different from the other granulocytic
dimly expressed on myelocytes and is gradually upregu- cells. They express CD22, but are negative for other B-cell
lated again as the granulocytic cells develop into seg- markers (39). An increase in the relative size of the baso-
mented neutrophils (Fig. 7D). Combination of this dy- philic cell population can be seen in the chronic and
namic marker with CD11b and CD16 in one accelerated phase of CML (48).
immunostaining reveals four sequential maturation stages Shifts in the relative distribution of the granulocytic
in the neutrophil development (Fig. 7A: stage IIV; Fig. subpopulations and in the immunophenotypic character-
7EG). CD11b and CD16 are initially expressed at low istics are seen in myeloid malignancies like AML and
levels, but their expression increases during the develop- myeloid dysplastic syndrome (MDS) (50,51). An increase
ment process, particularly in the last 2 stages (stages IV of immature granulocytic precursor cells or a reduction in
and V; Fig. 7E). well-differentiated granulocytes can be seen in both dis-
With the quadruple immunostaining 5 (CD16/CD13/ eases. The two immunostainings were not selected for the
CD45/CD11b), the myelo/monoblasts can be identied as identication of all sorts of immunophenotypic aberran-
CD16CD13CD45intermediateCD11b. They have an inter-
cies that can be expected in patients with AML or MDS,
mediate forward scatter (FSC) but still a low SSC and
but they are useful for a quick screening of BM for such
globally correspond to the CD34CD117CD45intermediate
abnormalities. First, immunostaining 3 (CD34/CD117/
CD13.33 cells from immunostaining 3 (Fig. 7). When the
CD45/CD13.33) will illustrate that most CD34 precursor
myeloblasts progress to the stage of promyelocytes, a
dramatic increase in SSC is seen (Fig. 7B,D). From this cells in MDS or AML BM belong to the myelo/monocytic
moment, the cells express CD15 and retain their high SSC. lineage, and not to the B-cell lineage, as they are CD117
In the subsequent maturation stages CD11b is acquired, and/or CD13.33. In addition, the intermediate CD45
followed by CD16 (Fig. 7EG). Note that the relatively expression in combination with SSC identies increased
high uorescence signal for CD16-uoroscein isothiocya- numbers of myelo/monoblast cells in both immunostain-
nate (FITC) of promyelocytes and myelocytes in Figure ings. Second, morphological abnormalities like hypo- or
7E,G has nothing to do with early CD16 expression but hypergranulation will be reected in reduced or increased
with the high autouorescence of these subpopulations in SSC values (Fig. 9). Third, in MDS an overexpression of
the FL1 (FITC) channel. CD13 and CD33 and a reduced expression of CD11b and
Like B-cell development, the staining patterns of immu- CD16 is frequently seen and consequently the CD13/
nostaining 5 for normal granulocytic differentiation in BM, CD16 and CD13/CD11b dot plots of immunostaining 5
are quite stable with regard to the immunophenotypic will show an aberrant expression prole of the granulo-
characteristics and relative distribution of the various sub- cytic cells (Fig. 9).

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

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 identied 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 identies development. The nucleated erythroid population can
two sequential differentiation stages of nonlysed nucle- be much more predominant in cases of a severe aplastic
ated erythroid cells in BM samples after red cell lysis (Fig. anemia or MDS, or during BM regeneration after drug-
10A). Cells of the erythroid lineage are generally identied induced BM suppression. In regenerating BM of children
by the gradual loss of CD45 (6). Only the CD117 CD71 treated for ALL, the erythroid cell population can occupy
erythroid-precursors and pro-erythroblasts do express up to 50% of the cells in the lysed BM sample (53). These
CD45 dimly. During maturation, the expression of the CD45CD71CD235a cells include both the nucleated
transferrin receptor CD71 slightly increases, where-after erythroid cells and the reticulocytes, which tend to be
CD235a (glycophorin A) is being expressed. The red cells more resistant to lysis when present in high numbers in
retain CD235a but lose CD71 together with loss of the young children (Fig. 11AC). Although shifts in the relative
nucleus (6,31). 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: CD117CD45lowCD71CD235a pro-erythroblasts (red
dots), CD117CD45neg-lowCD71CD235a erythroblasts (green) and CD117CD45CD71CD235a nonlysed erythrocytes (blue dots) can be

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BIOMED-1 concerted action investigation of minimal residual disease
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