Cytometry Part B (Clinical Cytometry) 72B:34–42 (2007)
Quantitative Analysis of the Expression
of Glycosylphosphatidylinositol-Anchored
Proteins During the Maturation
of Different Hematopoietic Cell
Compartments of Normal Bone Marrow
Pilar Marı́a Hernández-Campo, Julia Almeida, Sergio Matarraz, Marı́a de Santiago,
Marı́a Luz Sánchez, and Alberto Orfao
Servicio General de Citometrı́a and Departamento de Medicina and Centro de Investigación del Cáncer,
Universidad de Salamanca, Salamanca, Spain
Background: Glycosylphosphatidylinositol-anchored proteins (GPI-AP) are a heterogeneous group of
proteins deficiently expressed in patients with paroxysmal nocturnal hemoglobinuria. Up till now, no
study has been reported in which the exact patterns of expression of a large number of GPI-AP are quantitatively evaluated in normal bone marrow (BM) cells classified according to their lineage and maturation stage.
Methods: In the present study, we have quantitatively analyzed the expression of eleven different GPIAP (CD14, CD16, CD24, CD48, CD52, CD58, CD59, CD66b, CD87, CD109 and CD157) during maturation
of the neutrophil, monocytic, erythroid, lymphoid, basophil and plasmacytoid dendritic cells (DC) lineages
in normal BM as a frame of reference for the understanding of the abnormal patterns of expression of
GPI-AP observed in the BM of PNH patients.
Results: Our results show that expression of most GPI-AP varies during normal BM maturation, different
profiles being frequently observed depending on the cell lineage or the GPI-AP analyzed.
Conclusion: Overall, these results provide a detailed map GPI-AP expression during normal hematopoietic differentiation, which could serve as a basis for the identification and characterization of changes
occurring in PNH. q 2006 Clinical Cytometry Society
Key terms: GPI-anchored proteins; flow cytometry; normal bone marrow
Glycosylphophatidylinositol-anchored proteins (GPIAP) are a heterogeneous group of proteins that show different functions and patterns of expression in distinct
compartments of normal mature hematopoietic cells,
deficiently expressed in patients with paroxysmal nocturnal hemoglobinuria (PNH) (1–3). Despite the physiological and pathogenetic relevance of different GPI-AP in
PNH patients, the information about the patterns of
expression of these molecules during hematopoiesis
remains restricted to a relatively small number of GPI-AP
analyzed in a limited number of hematopoietic cell lineages. Accordingly, Terstappen et al. (4) have previously
reported that during normal hematopoietic maturation,
similar levels of both CD55 and CD59 are expressed on
CD34þþ/CD38/þ and CD34þþ/CD38þ/þ precursors, the
reactivity for both GPI-AP decreasing along the erythroid
maturation. Regarding myeloid cells, they detected upregulation of CD55 during both neutrophil and mono-
q 2006 Clinical Cytometry Society
cytic maturation; in contrast, expression of CD59 remained stable during maturation of both cell lineages. In
turn, during B-cell maturation, an increasingly high
expression of CD55 was observed in parallel with downregulation of CD59 (4). Other studies focusing on the
analysis of other GPI-AP, have shown that during neutrophil maturation CD24 and CD87 would become positive
Grant sponsor: Ministerio de Ciencia y Tecnologı́a; Grant number:
SAF 02-03096.
*Correspondence to: Alberto Orfao, Servicio General de Citometria,
Hospital Universitario de Salamanca, Paseo San Vicente, 58-182,
37007 Salamanca, Spain.
E-mail: [email protected]
Received 12 April 2006; Revision 9 June 2006; Accepted 20 July
2006
Published online 18 October 2006 in Wiley InterScience (www.
interscience.wiley.com).
DOI: 10.1002/cyto.b.20143
EXPRESSION OF GPI-ANCHORED PROTEINS ALONG THE MATURATION IN NORMAL BM
at the myelocyte and band maturation stages, respectively (5,6); in turn CD66b and CD16 would be
expressed at earlier stages of maturation, the highest
expression of CD16 being observed on bands and
mature neutrophils (7–11). CD14 expression would be
restricted to the latest stages of the maturation of normal
monocytic cells (12).
In contrast, to our knowledge no study has been
reported so far in which the expression of other GPI-AP
such as CD109, CD48, CD52, CD58, and CD157 has
been analyzed during maturation of normal hematopoietic cells. Moreover, analysis of CD55 and CD59 expression during hematopoiesis has been so far restricted to
the erythroid, neutrophil, monocytic, and B-cell lineages
(4), no information being available as regards their
expression in other hematopoietic cell compartments;
in addition, no flow cytometric quantitative analysis of
CD55, CD59, or CD14 and CD66b expression has been
reported in normal BM, aimed at comparing the reactivity for these proteins among different cell lineages as
well as during different maturation stages, in a standardized and reproducible way.
In the present study, we have quantitatively analyzed
the expression of a large number of GPI-AP (CD14,
CD16, CD24, CD48, CD52, CD55, CD58, CD59, CD66b,
CD87, CD109, and CD157) during normal maturation of
the neutrophil, monocytic, erythroid, lymphoid, basophil, and plasmacytoid dendritic cell (DC) lineages, as a
frame of reference for the understanding of the abnormal patterns of expression of GPI-AP observed in the BM
of PNH patients.
MATERIALS AND METHODS
Samples
Peripheral blood (PB) and bone marrow (BM) aspirate
samples from ten healthy adult volunteers (4 males and
6 females, aged 56 6 20 years) were obtained after
informed consent and placed into VacutainerTM tubes
containing EDTA as anticoagulant (Becton/Dickinson –
BD-Labware, Franklin Lakes, NJ).
35
Expression of each GPI-AP was studied using different
6-color combinations of MAb depending on the specific
cell lineage under study. Surface expression of CD55,
CD58, CD59, and CD109 was studied on CD34þ hematopoietic stem and progenitor cells counterstained with
CD45-allophycocyanin-Cy7 (APC.Cy7; clone: 2D1, BDB),
CD34-PECy7 (clone: 8G12; BDB), anti-HLADR-peridinin
chlorophyll protein (PerCP; clone: L243; BDB), and
CD117-APC (clone: YB5.B8; BDB). Reactivity for CD16,
CD24, CD55, CD58, CD59, CD66b, CD87, CD109, and
CD157 was assessed on neutrophil lineage cells counterstained for CD45-APC.Cy7, CD34-PECy5 (clone: 581;
IOT), CD11b-PECy7 (clone: ICRF44; BDB), and CD13APC (clone: WM15; BDB). In turn, for the analysis of the
expression of CD14, CD48, CD52, CD55, CD58, CD59,
CD87, CD109, and CD157 on monocytic cells BM samples were stained simultaneously for the above mentioned markers and both CD64-PECy5 (clone: 10.1; Caltag) and CD36-APC (clone: CB38; BDB) in addition to
CD45-APC.Cy7 and CD34-PECy7. Reactivity for CD55,
CD58, and CD59 was evaluated on red cells simultaneously stained for CD71-PECy5 (clone: M-A712; BDB), glycophorin A-APC (clone: GA-R2; BDB), CD45-APC.Cy7,
and CD34-PECy7. Additionally, the coexpression of CD55
and CD59 was analyzed on basophils and plasmacytoid
DC stained with CD123-PECy5 (clone: 9F5; BDB), antiHLADR-APC (clone: L243; BDB), CD45-APC.Cy7, and
CD34-PECy7. Finally, expression of CD24, CD48, CD52,
CD55, and CD59 was also analyzed in normal BM B-cell
precursors (BCP) counterstained with the CD19-PerCPCy5.5
(clone: SJ25C1; BDB), CD38-APC (clone: HB7; BDB), CD45APC.Cy7, and CD34-PECy7.
For all stainings described above a well-established
stain-and-then-lyse protocol (13,14) was used, except for
those combinations of MAb which included the CD55,
CD58, and CD59 reagents; for these latter stainings a
previously described lyse-wash-and-then-stain protocol
was employed (13) in order to lyse non-nucleated red
cells prior to the staining procedure, and then avoid
their bind to these MAb. Expression of CD55, CD58, and
CD59 on red cells from PB was evaluated using a nonlyse-non-wash protocol, previously described in detail
(13).
Sample Preparation
The expression of GPI-AP was studied with the following monoclonal antibody (MAb) reagents: CD14 conjugated with fluorescein isothiocyanate (FITC) (clone:
MfP9), CD48-FITC (clone: TÜ145), CD55-phycoerythrin
(PE) (clone: IA10), CD58-PE (clone: L306.4), CD87-PE
(clone: VIM5), and CD109-PE (clone: TEA 2/16) were
purchased from BD Biosciences (BDB; San Jose, CA);
CD16-FITC (clone: 3G8), CD157-FITC (clone: RF3), and
CD24-PE (clone: ALB9) were obtained from Immunotech
(IOT; Marseille, France); CD59-FITC (clone: VJ.1/2) was
obtained from Immunostep S.L. (Salamanca, Spain);
CD66b-FITC (clone: 80H3) was from Cytognos S.L. (Salamanca, Spain) and CD52-PE (clone: CF1D12) was purchased from Caltag Laboratories (San Francisco, CA).
Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b
Flow Cytometry Data Acquisition and Analysis
Data acquisition was performed immediately after
completion of sample preparation using a FACSAria flow
cytometer (BDB) equipped with the FACSDivaTM software programme (BDB). Information on a total of 106
events corresponding to all nucleated cells present in
the sample was acquired for each staining.
For data analysis the FACSDivaTM software programme
(BDB) was used. The identification of the different maturation-associated cell subsets analyzed was performed as
shown in Figure 1 (panel I). Note that, although it is
known that there is not an exact correlation between
the immunophenotypic and the morphological stages,
the terms used to identify the different neutrophil and
36
HERNANDEZ-CAMPO ET AL.
FIG. 1. Panel I: Representative bivariate dot plots and histograms illustrating how the different maturation-associated cell subsets analyzed in this
study were identified for the analysis of the expression of GPI-AP, according to previous studies (12). Panels A, B, and C show how CD34þ early neutrophil lineage cells (red events) and CD34þ early monocytic precursors (blue events) were identified, respectively. Panels D and E show how CD34/low
myeloblasts (blue events), promyelocytes (violet events), myelocytes/metamyelocytes (green events), and bands/mature neutrophils (red events) were
identified. The strategy used for the identification of both promonocytes CD64þ/CD36low (blue events) and CD64þ/CD36þ (red events) monocytic cells is
illustrated in panels F and G. Panels H and I show how CD34þ early erythroid precursors (CD34þ/CD19/CD45dim/SSClow, red events) were identified.
Identification of both the CD71high and CD71þ subpopulations of erythroblasts is illustrated in panels J and K. Panels L and M show how CD34þ precursors (black events) and CD123high cells (violet events) were selected; identification of CD34þ cells (grey events), plasmacytoid DC precursors (green
events), plasmacytoid DC (yellow events), basophil precursors (blue events), and basophils (red events) is shown in panel N. Panels O and P illustrate
how CD34þ (blue events) and CD34 (II, red events) B-cell precursors as well as mature B-cells (green events) were identified. Panel II: Representative
bivariate dot plots and histograms of GPI-AP expression on different maturation associated cell-subsets present in BM samples from normal individuals.
monocytic maturation stages have been based on conventional morphological designations. The mean fluorescence intensity (MFI; expressed in relative linear units
scaled from 0 to 262,144) was recorded for each GPI-AP
after specifically gating for each BM cell subset of interest. Evaluation of the antibody binding capacity (ABC)
for those antigens recognized by PE- and FITC-conjugated IgG MAb was performed using the QuantiBRITE
PE kit (BDB) (15) and the QuantumTM Simply Cellular
kit (Bangs Laboratories, Fisher, IN) (16), respectively, following the recommendations of the manufacturer and
using their dedicated software programmes. ABC units
could not be calculated for the CD48 antigen since it
was a FITC-conjugated mouse IgM MAb. Results of nega-
tive controls did not affect the ABC or MFI values in the
cell lineages analyzed.
Statistical Methods
The mean value 6 standard deviation, median, range,
the 25th and 75th percentiles and the 95% confidence
intervals were calculated for all parameters using the
SPSS 11.0 software programme (SPSS, Chicago, IL). The
statistical significance of the differences observed between the distinct subsets of cells identified was calculated using the paired Wilcoxon non-parametric test.
P-values <0.05 were considered to be statistically significant.
Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b
EXPRESSION OF GPI-ANCHORED PROTEINS ALONG THE MATURATION IN NORMAL BM
37
FIG. 2. Expression of CD55, CD58, CD59, CD109, CD24, CD66b, CD157, CD16, and CD87 during normal BM maturation of cells from the neutrophil lineage. Results are expressed in antibody binding capacity (ABC) units. Notched-boxes represent 25th and 75th percentile values; the line in
the middle and vertical lines correspond to the median value and both the 10th and 90th percentiles, respectively. * P < 0.05, early CD34þ neutrophil precursors vs.CD34/low myeloblasts, promyelocytes, myelocyte/metamyelocytes and bands/mature neutrophils; # P < 0.05, early CD34þ neutrophil precusors vs.CD34/low myeloblasts and promyelocytes; P < 0.05, CD34/low myeloblasts vs.promyelocytes, myelocyte/metamyelocytes, and
bands/mature neutrophils; ? P < 0.05, myeloblasts vs.promyelocytes; D P < 0.05, CD34/low myeloblasts vs.myelocyte/metamyelocytes; ! P <
0.05, CD34/low myeloblasts vs.bands/mature neutrophils; * P < 0.05, promyelocytes vs.myelocytes/metameylocytes and bands/mature neutrophils;
& P < 0.05, promyelocytes vs.bands/mature neutrophils; ! P < 0.05, myelocytes/metamyelocytes vs.bands/mature neutrophils.
RESULTS
Expression of GPI-AP During Neutrophil Maturation
From all GPI-AP studied, CD55, CD58, CD59, and
CD109 were the only proteins constantly expressed
along all the different stages of maturation of the neutrophil cell lineage. As illustrated in Figure 2, no significant
differences were detected for CD59 during the different
stages of maturation of the neutrophil lineage analyzed.
In contrast, expression of both CD55 and CD109 significantly decreased from the early CD34þ neutrophil precursors to the promyelocyte stage (P < 0.05) to show a
later increase along the maturation towards mature neutrophils (P < 0.05). In turn, CD58 expression increased
early (P ¼ 0.01 for CD34/low myeloblasts vs.early
CD34þ neutrophil precursors), its expression remaining
stable till the metamyelocyte stage and significantly
decreasing thereafter (P ¼ 0.03 for bands/mature neutrophils vs.CD34/low myeloblasts and promyelocytes).
Expression of CD24, CD66b, and CD157 (Fig. 2) was
absent on the early CD34þ neutrophil precursors,
becoming positive at the stage of CD34/low myeloblasts.
Then expression of both CD24 and CD66b increased
until the myelocyte stage (P < 0.01), decreasing thereafter to less than half on bands/mature neutrophils.
In turn, CD157 was found to be expressed at lower
levels throughout the neutrophil maturation, its reactiv-
Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b
ity slightly increasing (P < 0.001) at the later stages of
maturation, from the promyelocyte stage on. CD16 was
expressed on myelocytes/metamyelocytes, its reactivity
increasing on the bands/mature neutrophils (P ¼ 0.001),
while CD87 only became detectable on this latter cell
population. The qualitative patterns of expression observed for these markers were similar in the 10 BM samples analyzed.
Expression of GPI-AP During Monocytic Maturation
CD55, CD58, CD59, and CD109 were found to be
present throughout the different stages of the monocytic
maturation identified (Fig. 3). Expression of both CD55
and CD59 on CD36low monocytic cells was similar to
that observed on the early CD34þ monocytic precursors
(P > 0.05), but it significantly increased on CD36high
monocytic cells (P ¼ 0.01). In turn, the patterns of
expression of CD58 and CD109 revealed a progressively
higher reactivity for both markers along the monocytic
maturation associated with a significantly higher reactivity (P 0.03) on mature monocytes as compared to
monocytic precursors.
Reactivity for CD14, CD87, CD157 (Fig. 3A), and
CD48 (Fig. 3B) was absent on early CD34þ monocytic
precursors becoming clearly positive only on the CD36low
stage of maturation; on the more mature CD36high mono-
38
HERNANDEZ-CAMPO ET AL.
FIG. 3. Expression of CD55,
CD58, CD59, CD109, CD14,
CD52, CD157 (panel A), and CD48
(panel B) during maturation of
monocytic cells in the BM. Results
are expressed either as antibody
binding capacity (ABC) units (panel
A) or as mean fluorescence intensity
(MFI) values (arbitrary relative linear fluorescence units scaled from
0 to 262,144) (panel B). Notchedboxes represent 25th and 75th percentile values; the line in the middle and vertical lines correspond to
the median value and both the
10th and 90th percentiles, respectively. * P < 0.05, early CD34þ
monocytic precursors vs. both
and
CD64þ/
CD64þ/CD36low
CD36high monocytic cells; P <
0.05, early CD34þ monocytic precursors vs.CD64þ/CD36high monocytic cells; & P < 0.05, CD64þ/
CD36low vs.CD64þ/CD36high monocytic cells.
cytic cells all markers being strongly expressed (P ¼ 0.01
for CD36high vs. CD36low monocytic cells). Again, the
qualitative patterns of expression of these markers
throughout the monocytic maturation were similar in
all normal BM samples analyzed.
Expression of the GPI-AP During Maturation of Erythroid,
Basophil, and Plasmacytoid Dendritic Cells
As shown in Figure 4 (panel A), in all BM studied,
expression of both CD58 and CD59 was transiently
increased on CD71high erythroblasts (P 0.02) decreasing towards the initial levels on CD71þ erythroblasts,
the reactivity for this marker on the latter stage being
similar to that observed on erythrocytes from PB. In contrast, reactivity for CD55 showed a progressive decrease
from the early CD34þ erythroid precursors towards the
red cells from PB (P < 0.01).
As illustrated in Figure 4 (panel B), no significant differences were observed on the expression of CD55
along the maturation of plasmacytoid DC while the reactivity for CD59 significantly decreased in the more
mature plasmacytoid DC (P < 0.03). In turn, maturation
into basophils was associated with a significantly lower
expression of CD59 (P < 0.05) and a higher reactivity
for CD55 (P < 0.03) (Fig. 4C).
Expression of GPI-AP During Maturation of B-Cells
In all normal BM samples analyzed, the expression of
GPI-AP during B-cell maturation was homogeneous within
each differentiation stage. Accordingly, CD24, CD52, CD55,
Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b
EXPRESSION OF GPI-ANCHORED PROTEINS ALONG THE MATURATION IN NORMAL BM
FIG. 4. Expression of GPI-AP during maturation
of erythroid cells (panel A), plasmacytoid DC (panel
B), and basophils (panel C) in normal BM. Results
are expressed in antibody binding capacity (ABC)
units. Notched-boxes represent 25th and 75th percentile values; the line in the middle and vertical
lines correspond to the median value and both the
10th and 90th percentiles, respectively. * P <
0.05, early CD34þ erythroid precursors vs.CD71high
erythroblasts; P < 0.05, early CD34þ erythroid
precursors vs.CD71þ erythroblasts; D P < 0.05,
early CD34þ erythroid precursors vs.PB erythrocytes; & P < 0.05, CD71high erythroblasts
vs.CD71þ erythroblasts and PB erythrocytes; * P
< 0.05, CD71þ erythroblasts vs.PB erythrocytes
(Panel A). # P < 0.05, plasmacytoid DC precursors
vs.plasmacytoid DC (Panel B); ? P < 0.05, Basophil precursors vs.basophil (Panel C).
Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b
39
40
HERNANDEZ-CAMPO ET AL.
FIG. 5. Expression of the CD24,
CD52, CD55, CD59 (panel A), and
CD48 (panel B) GPI-AP during normal BM B-cell maturation. Results
are expressed either in antibody
binding capacity (ABC) units (panel
A) or as mean fluorescence intensity
(MFI) values expressed in fluorescence channel units (arbitrary relative linear units scaled from 0 to
262,144) (panel B). Notched-boxes
represent 25th and 75th percentile
values; the line in the middle and
vertical lines correspond to the median value and both the 10th and
90th percentiles, respectively. * P
< 0.05, CD34þ/CD38þ/CD19þ Bcell precursors vs.both CD34/
CD38þ/CD19þ B-cell precursors
and CD34/CD38/CD19þ mature
B-cells; * P < 0.05, CD34þ/
CD38þ/CD19þ B-cell precursors
vs.CD34/CD38/CD19þ mature Bcells; & P < 0.05, CD34/CD38þ/
CD19þ B-cell precursors vs.CD34/
CD38/CD19þ mature B-cells.
and CD59 were positive throughout all BM B-cell maturation stages. Expression of CD55 increased from CD34þ to
CD34 B-cell precursors (P < 0.05), its levels on this later
compartment being similar to those observed on mature
B-cells (P > 0.05). In contrast, no significant differences
were observed on CD59 expression along the B-cell maturation. Regarding CD24, it was strongly expressed on
both CD34þ and CD34 B-cell precursors, its levels significantly decreasing on mature B-lymphocytes (P ¼ 0.02).
In turn, expression of CD52 was transiently decreased on
CD34 B-cell precursors (P ¼ 0.01), reaching the highest
levels on the more mature B-lymphocytes (P < 0.05).
Finally, expression of CD48 was only detectable after
maturation of CD34þ into CD34 B-cell precursors, its
expression significantly increasing thereafter (P ¼ 0.03)
(Fig. 5).
DISCUSSION
Differentiation and maturation of BM hematopoietic
cells can be monitored through the occurring immunophenotypic-changes. Up till now, the reactivity for a
high number of proteins has been studied along the
maturation of different BM hematopoietic cell lineages
(7,12,17–19). Despite this, the information currently
Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b
EXPRESSION OF GPI-ANCHORED PROTEINS ALONG THE MATURATION IN NORMAL BM
available about the patterns of expression of GPI-AP during normal hematopoiesis is limited to a few cell lineages and to a restricted number of markers. In addition,
no study has quantitatively evaluated the patterns of
expression of GPI-AP in normal BM cells using objective,
reproducible, and standardized flow cytometric methods.
To our knowledge, this is the first report in which
expression of a large number of GPI-AP during maturation of different hematopoietic cells in normal BM is analyzed using a well-standardized quantitative approach.
Previous studies have investigated the expression of
CD55, CD59, CD24, CD87, CD16, and CD66b during
normal maturation of neutrophil cells in a qualitative or
semiquantitative way (4–6,8,9,11). Our results confirm
and extend previous results showing that quantitatively
the expression of GPI-AP varies throughout the neutrophil maturation. We have observed that, not only CD55
and CD59, but also CD58 and CD109, are expressed during the whole maturation of neutrophil cells from the
early neutrophil precursors to mature cells, although the
pattern of expression of each antigen is different. Thus,
the expression of CD55 and CD109 decreases from the
early precursors till the intermediate stages of maturation, increasing thereafter. In contrast, CD58 followed an
inverse pattern, while no significant changes in the reactivity for CD59 were detected. Previous studies indicate
that the myelocyte is the first cell within the neutrophil
lineage expressing CD24 (5); however, our results indicate that expression of CD24 as well as that of CD66b
and CD157 is already detectable on CD34/low myeloblasts, the three markers reaching their highest levels of
expression on normal BM myelocytes, decreasing thereafter with maturation. Such discrepancies could be due
to the use of less sensitive fluorochromes, MAb reagents,
and instruments. As expected (14), the expression of
CD16 was detectable on metamyelocytes, reaching the
highest levels on bands/mature neutrophils, while expression of CD87 was only detectable at this latter stage
of neutrophil maturation, as expected (6).
Interestingly, as described for the neutrophil lineage,
CD55, CD59, CD58, and CD109 were also found to be
present throughout the different stages of maturation of
monocytic cells. Previous studies have reported up-regulation of CD55 with stable amounts of CD59 during
monocytic maturation (9). However, our results clearly
show that the expression of both antigens significantly
increases on mature monocytes as compared to the
more immature monocytic cells. To our knowledge, this
is the first report in which expression of CD58, CD109,
CD52, CD87, and CD157 is analyzed during monocytic
maturation. Our results show a similar pattern of expression for CD58 and CD109 with progressively higher
amounts of both proteins detected per cell in the more
advanced stages of the monocytic maturation. In turn,
CD52, CD87, CD157, and CD14 were absent on the earliest monocytic precursors identified; while the former
three markers appeared on CD36low monocytic cells and
were expressed at higher levels on the more mature
CD36high monocytes, expression of CD14 could only be
Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b
41
clearly detected at this latter stage of maturation of the
monocytic cell lineage (12).
Regarding red cells, our results confirm previous
observations, suggesting that the expression of both
CD55 and CD59 varies along the erythroid maturation
(9). Accordingly, CD55 expression progressively decreased from the early erythroid precursors toward the
more mature red cells; in contrast, reactivity for CD59
peaked at CD71high erythroblasts, decreasing thereafter.
In the present study, expression of CD58 was also analyzed in this cell lineage, displaying a similar pattern of
expression to that of CD59.
To our knowledge, no previous studies analyzing the
expression of GPI-AP on the basophil and plasmacytoid
DC lineages have been reported. In this regard, our
results show the absence of statistically significant
changes in the expression of CD55 throughout the maturation of plasmacytoid DC, while CD59 significantly
decreases in the more mature plasmacytoid DC as compared to their precursors. A similar pattern of expression
of CD59 was observed on the basophil cell lineage but
here in association with a significantly higher reactivity
for CD55 on mature basophils as compared to their precursors.
Regarding B-cells, we confirmed previous observations
showing a decreased expression of CD24 (20) and an
increased reactivity for CD55 (4) on mature B-cells as
compared to the overall population of BCP. However, in
contrast to the observations of Terstappen et al, expression of CD59 remained constant throughout the different
B-cell maturation stages. In addition, CD55 expression
was clearly lower in CD34þ as compared to CD34 BCP.
CD48 has been considered one of the most useful
markers for the identification of PB lymphocytes in PNH
(21,22). However, the pattern of expression of this
marker during normal B-cell maturation in the BM
remains unclear. According to our results, CD48 expression can be first detected on CD34 B-cell precursors,
its levels increasing on the more mature CD45high/
CD38/low B-cell compartment. CD52 is a lymphocyteassociated protein (21) whose quantitative expression
along the maturation of normal BM B-cells has not been
reported so far. Our results show that CD52 is expressed
along all B-cell maturation stages, mature B-cells showing
the highest levels of expression for this protein.
The above mentioned variations in the expression of
GPI-AP could be related to the specific function of each
protein. Thus, complement-regulatory proteins such as
CD55 and CD59 are expressed in all different hematopoietic cell lineages from early precursors to mature
cells to protect them from autologous, complementmediated injury (9). According to our results, the expression of both markers is up-regulated in most cell lineages, except on erythroid cells, for which, the lowest
expression is observed on PB erythrocytes. In turn,
other GPI-AP related to the immune response such as
CD14 (LPS receptor) (22) and CD16 (Fcg receptor III)
(10,23) appear at the later stages of the monocytic and
neutrophil maturation, respectively.
42
HERNANDEZ-CAMPO ET AL.
Of note, quantitative analyses of individual protein cell
surface levels were measured in this study to provide an
extra level of standardization and to make our findings
reproducible an easier way. However, preliminary studies
performed in BM samples from PNH patients show that
qualitative analyses of the above mentioned markers are
a sufficiently robust baseline for discriminating between
normal and PNH cells within the different cell subsets
(data not shown). Analysis of a large series of patients
would be required to confirm these preliminary observations. The expression of GPI-AP was also analyzed in BM
samples from patients with myelodysplastic syndromes
(MDS), no significant differences being detected compared to normal BM (data not shown).
In the present study we have analyzed BM samples
from normal healthy volunteers. These analyses are
expected to provide a frame of reference for the study
of BM samples from different disease conditions, particularly PNH. However, it should be noted that although
the analysis of PNH BM samples may provide additional
information about the pathogenesis of the disease, at the
same time it might be of greater utility than the study of
PB in the differential diagnosis of uncommon cases, in
most occasions, analysis of PB would allow diagnosis of
PNH in clinical practice.
In summary, our results show that expression of most
GPI-AP varies during normal BM maturation different
profiles being frequently observed depending on the specific cell lineage or GPI-AP analyzed. These results provide a detailed map of GPI-AP expression during normal
hematopoietic differentiation which could serve as a basis for the identification and characterization of changes
occurring in PNH.
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Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b