Prolonged Thrombocytopenia Following Allogeneic Hematopoietic Stem Cell Transplantation and Its Association with a Reduction in Ploidy and an Immaturation of Megakaryocytes

Abstract

Prolonged thrombocytopenia is a frequent complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, its pathogenesis has remained obscure. In the present study, we used flow cytometry to determine the frequency of bone marrow megakaryocytes (MKs) and MK ploidy distributions in allo-HSCT recipients with or without prolonged thrombocytopenia (n = 32 and 27, respectively) and healthy volunteers (n = 13). In addition, the expression of c-Mpl in MKs was measured. The results indicate that the proportions of MKs in marrow mononuclear cells or the percentages of CD110(+) MKs in total MKs did not significantly differ between the 3 groups; however, in a comparison of nonthrombocytopenic allo-HSCT recipients to healthy volunteers, the allo-HSCT patients who had prolonged thrombocytopenia exhibited significant shifts toward low ploidy cells (left shift), which were accompanied by a marked increase in ≤ 8N cells (P = .036 and P < .001, respectively) and significant decreases in 16N cells (P < .001 and P < .001, respectively) and ≥ 32N cells (P = .01 and P <.001, respectively). These results indicate that there were more immature MKs in allo-HSCT recipients who had prolonged thrombocytopenia, in comparison to nonthrombocytopenic allo-HSCT recipients and healthy volunteers. We conclude that prolonged thrombocytopenia and slow platelet engraftment after allo-HSCT may be related to a reduction in ploidy and an immaturation of MKs.

Full text

Prolonged Thrombocytopenia Following Allogeneic
Hematopoietic Stem Cell Transplantation and Its
Association with a Reduction in Ploidy and an
Immaturation of Megakaryocytes
Xiaohui Zhang,
1,
*Haixia Fu,
1,
*Lanping Xu,
1
Daihong Liu,
1
Jianzhong Wang,
2
Kaiyan Liu,
1
Xiaojun Huang
1
Prolonged thrombocytopenia is a frequent complication after allogeneic hematopoietic stem cell transplanta-
tion (allo-HSCT); however, its pathogenesis has remained obscure. In the present study, we used flow
cytometry to determine the frequency of bone marrow megakaryocytes (MKs) and MK ploidy distributions
in allo-HSCTrecipients with or without prolonged thrombocytopenia (n 532 and 27, respectively) and healthy
volunteers (n 513). In addition, the expression of c-Mpl in MKs was measured. The results indicate that the
proportions of MKs in marrow mononuclear cells or the percentages of CD110
1
MKs in total MKs did not
significantly differ between the 3 groups; however, in a comparison of nonthrombocytopenic allo-HSCTrecip-
ients to healthy volunteers, the allo-HSCT patients who had prolonged thrombocytopenia exhibited significant
shifts toward low ploidy cells (left shift), which were accompanied by a marked increase in #8N cells (P5.036
and P\.001, respectively) and significant decreases in 16N cells (P\.001 and P\.001, respectively) and $32N
cells (P5.01 and P\.001, respectively). These results indicate that there were more immature MKs in allo-
HSCT recipients who had prolonged thrombocytopenia, in comparison to nonthrombocytopenic allo-HSCT
recipients and healthy volunteers. We conclude that prolonged thrombocytopenia and slow platelet engraft-
ment after allo-HSCT may be related to a reduction in ploidy and an immaturation of MKs.
Biol Blood Marrow Transplant 17: 274-280 (2011) Ó2011 American Society for Blood and Marrow Transplantation
KEY WORDS: Thrombocytopenia, Megakaryocyte, Ploidy, Allogeneic hematopoietic stem cell transplantation
INTRODUCTION
Prolonged thrombocytopenia, which is defined as
a recovery of all peripheral blood cell lines aside from
consistently low platelet counts after transplantation
for more than 3 months, is a frequent complication
after allogeneic hematopoietic stem cell transplantation
(allo-HSCT). Severe thrombocytopenia necessitates
platelet transfusion for the management and prevention
of bleeding and influences the therapeutic effects
and prognosis of transplantation. Kim et al. [1] have
identified increased thrombocytopenia-related mortal-
ity and infection rates in thrombocytopenia patients af-
ter transplantation, wherein low platelet counts on the
60th day after transplantation was an independent risk
factor for poor allo-HSCT patient prognosis [2].Addi-
tionally, other studies have described poor prognoses in
patients with thrombocytopenia 90 days after allogeneic
bone marrow transplantation [3,4].
The major causes of thrombocytopenia include ac-
celerated peripheral platelet destruction by antiplatelet
antibodies and the insufficient production of platelets
from marrow megakaryocytes (MKs). In previous stud-
ies, both increased platelet turnover and impaired
thrombopoiesis have been reported to be involved in
the development of prolonged thrombocytopenia after
HSCT, wherein the latter mechanism plays a predomi-
nant role [5,6]; however, until recently, little has been
understood regarding the development and maturation
of the immediate precursors of platelets, which are the
marrow MKs, in post-allo-HSCT patients.The primary
feature of MKmaturation is the development of asingle,
From the
1
Peking University People’s Hospital, Peking University
Institute of Hematology, Beijing, People’s Republic of China;
and
2
Peking University First Hospital, Department of Clinical
Laboratory, Beijing, People’s Republic of China.
Financial disclosure: See Acknowledgments on page 279.
Xiaohui Zhang and Haixia Fu share the first authorship.
*The authors contributed equally to this article.
Correspondence and reprint requests: Xiaojun Huang, MD, Peking
University People’s Hospital, Peking University Institute of He-
matology, Beijing 100044, P.R. China (e-mail: xjhrm@medmail.
com.cn).
Received July 7, 2010; accepted September 12, 2010
Ó2011 American Society for Blood and Marrow Transplantation
1083-8791/$36.00
doi:10.1016/j.bbmt.2010.09.007
274

large, lobulated, and polyploid nucleus. Mature MKs
cease to proliferate but continue to increase their
DNA content without undergoing thelate stages of mi-
tosis [7-9]. To our knowledge, no study has evaluated the
ploidy pattern in MKs in post-allo-HSCT patients.
C-Mpl (CD110), which is expressed on the surfaces
of MKs and MK precursors, is a receptor for thrombo-
poietin (TPO). TPO has been identified to be a key
cytokine for megakaryogenesis and thrombopoiesis be-
cause it binds to c-Mpl (CD110). Yamazaki et al. [6]
have found that plasma TPO was significantly increased
in HSCT recipients with thrombocytopenia in compar-
ison to those without; however, no study has focused on
c-Mpl (CD110) expression in MKs in post-allo-HSCT
patients.
The aim of the present study was to evaluate
whether the ploidy distribution pattern in MKs and
the level of c-Mpl expression on the surface of
MKs in allo-HSCT patients with prolonged thrombo-
cytopenia differed from those in allo-HSCT patients
without prolonged thrombocytopenia or healthy
volunteers.
PATIENTS AND METHODS
Patients and Controls
Prolonged thrombocytopenia was defined as
a platelet count #80 10
9
/L for more than 3 months
after HSCT, recovery of all other cell counts, and no
apparent cause for thrombocytopenia, such as engraft-
ment failure, recurrence of the underlying malignancy,
microangiopathy, or drugs. We studied 32 allo-HSCT
recipients who had prolonged thrombocytopenia. As
a control, we selected 27 allo-HSCT recipients who
did not have prolonged thrombocytopenia after day
90 and who had recovered all other cell counts. To
minimize the potential influence of the length of
time after allo-HSCT, all of the enrolled patients
had their bone marrow tested approximately 3 months
after allo-HSCT (median 188 days, range: 183 to
1120 days). Patients were excluded if they died, had
disease recurrence within 90 days of transplantation,
or if they initially failed neutrophil engraftment .0.5
10
9
/L. Clinical characteristics were matched be-
tween the study and control groups when possible.
All of these patients underwent allo-HSCT for various
hematologic malignancies or nonmalignancies be-
tween October of 2008 and May of 2009 at Peking
University People’s Hospital.
Bone marrow samples from 13 healthy volunteers
were used as healthy control subjects. The 13 healthy
controls consisted of 6 males and 7 females, and their
ages ranged from 16 to 51 years (median: 28 years).
Bone marrow samples were obtained after the pa-
tients and control subjects gave their written informed
consent. The institutional review board of the Peking
University Institute of Hematology and the ethics
committee of Peking University People’s Hospital
approved this study.
allo-HSCT Procedure
The conditioning therapy for HLA-mismatched
and unrelated matched HSCT patients included
modified BUCY2 plus ATG (thymoglobulin), which
consisted of intravenous cytarabine (4 g/m
2
/day) on
days –10 to –9, intravenous busulfan (3.2 mg/kg/day)
on days –8 to –6, intravenous cyclophosphamide
(1.8 g/m
2
/day) on days –5 to –4, oral Me-CCNU
(250 mg/m
2
) once on day –3; and intravenous antithy-
mocyte globulin (ATG) (2.5 mg/kg/day; Sang Stat,
Lyon, France) on days –5 to –2. In matched sibling
transplantations, patients received a regimen that was
identical to that of HLA-mismatched patients without
ATG except they orally received hydroxycarbamide
(80 mg/kg) on day –10 and a lower dose of cytarabine
(2 g/m
2
/day) on day –9. The transplanted grafts were
both granulocyte colony-stimulating factor-mobilized
peripheral blood stem cells (PBSCs) and bone marrow
(BM) cells from matched sibling or HLA-mismatched
sibling donors or PBSCs from unrelated donors. Post
allo-HSCT, filgrastim rh-granulocyte-colony stimu-
lating factor (rhG-CSF) was subcutaneously adminis-
tered to HLA-mismatched and unrelated matched
HSCT patients at 5 mg/kg/day from day 16 after trans-
plantation until the neutrophil count reached 0.5 10
9
cells/L for 3 consecutive days; however, in matched sib-
ling transplantations, filgrastim (rhG-CSF) was not
used post-allo-HSCT.
All patients received cyclosporine A (CsA), myco-
phenolate mofetil (MMF), and short-term methotrexate
for graft-versus-host disease (GVHD) prophylaxis
[10,11]. Methotrexate (MTX) was intravenously
administered at 15 mg/m
2
on day 11, at 10 mg/m
2
on
days 13and16 after all of the transplantations, and
again at 10 mg/m
2
on day 111 after HLA-mismatched
and unrelated matched HSCT. MMF was discontinued
upon engraftment after matched sibling HSCT,
whereas in the patients with HLA-mismatched and un-
related matched HSCT, MMF was tapered from 1 g/day
to 0.5 g/day on day 30 and was discontinued over days 45
to 60 based on the presence or absence of severe GVHD,
infectious diseases, or risk of relapse. Acute and chronic
GVHD (aGVHD, cGVHD) were defined according to
published criteria [12,13].
All allo-HSCT patients were screened pretrans-
plantation for cytomegalovirus (CMV) serostatus.
Weekly polymerase chain reaction (PCR) (Roche,
Amplicor, Indianapolis, IN, USA) was employed to
survey CMV reactivation in the blood from the time
of transplantation to 100 days afterward.
Biol Blood Marrow Transplant 17:274-280, 2011 275MKs Ploidy and Platelet Recovery

Bone marrow aspiration and cytogenetic studies
were performed 1, 2, and 3 months after transplanta-
tion to assess engraftment. HLA DNA typing and
PCR DNA fingerprinting (short tandem repeat) were
used for donor chimerism detection. For each patient,
at least 2 methods were used to confirm donor
chimerism.
Evaluation and Definitions for allo-HSCT
Patients
Engraftment was documented by increasing neutro-
phil and platelet counts that were unsupported by trans-
fusions. Neutrophil engraftment after transplantation
was defined as an absolute neutrophil count (ANC) in
excess of 500/mL for 3 consecutive days. The first of
these 3 consecutive days was considered to be the day
of engraftment. The day of platelet engraftment was de-
fined as the first day of 7 consecutive days in which the
patient had a platelet count of .20,000/mL and did
not receive a platelet transfusion. The diagnosis of graft
failure was made if persistently falling peripheral blood
counts and progressive bone marrow hypoplasia
(\25% cellularity) occurred after documented engraft-
ment; occurred in the absence of leukemic relapse,
drug toxicity, or infection; and persisted for at least 14
days. The day of graft failure was assigned to the day
that the ANC decreased to \500/mL.
Preparation of Bone Marrow Cells
Six milliliters of fresh bone marrow, which had been
collected in EDTA tubes, were processed within 6 hours
after collection and stored at room temperature. MKs
were analyzed in the fractionated marrow (enriched for
MKs to hasten the analysis). Fractionation was per-
formed by separating marrow cells over a discontinuous
Percoll gradient at a density of 1.060 g/mL. This proce-
dure yielded an approximate 15-fold enrichment with
recovery of at least 98% of recognizable MKs [14].
MK Staining
Tomer et al. [15,16] observed that although von
Willebrand factor (vWF) and CD41a as markers for
the detection of MKs exhibited similar overall
sensitivities and specificities, vWF was a better flow
cytometric (FCM) marker for distinguishing 2N/4N
MKs from other bone marrow nucleated cells.
Therefore, the vWF marker was used herein to detect
MKs. The cell count of the enriched cells was adjusted
to 2-3 10
6
/mL via a phosphate buffer solution (PBS)
and the cells werestained with anti-CD110 that was con-
jugated to allophycocyanin (APC) (BDIS, US) for sur-
face antigen staining according to the manufacturer’s
instructions. To measure the MK ploidy distribution,
the enriched marrow cells were simultaneously labeled
with fluorescein conjugated-mAb to VWF and stained
with propidium iodide (PI) for DNA. The marrow cells
were initially incubated in the presence of 0.025%
Triton X-100 for permeabilization and then labeled
with vWF-FITC mAbs (AbD Serotec, UK). After im-
munofluorescence staining, the cells were treated for
30 minutes with PI (a final concentration of 50 mg/mL)
and RNAse (a final concentration of 100 U/mL) at
37C for DNA quantification prior to flow cytometric
analysis. Aliquots of the marrow cell suspension were
also incubated under identical conditions with isotype-
matched mAbs and used as the control cell population.
Flow Cytometric Analysis
Flow cytometry was performed using a FACScan
flow cytometer (Becton Dickinson, BD Biosciences,
San Jose, CA, USA), which was equipped with a 488-
nm argon laser, using a low flow rate. Cells that ex-
pressed vWF and exhibited sizes that were larger than
the main marrow cell population were considered to
be MKs. To adequately analyze the ploidy distribution,
1000 to 3000 MKs were analyzed in each sample. The
acquisition rate was limited to 1000 cells/s so as to en-
hance resolution. Bidimensional plots of immunofluo-
rescence versus FSC or DNA-fluorescence were used
to establish the location of the desired cell population.
The ploidy distribution was determined by setting
markers at the nadirs between peaks using the 2N and
4N peaks of whole marrow cells as internal reference
standards. The expression of CD110 on the surface of
MKs was determined on the basis of their distinct im-
munofluorescence at levels in excess of that of control
cells labeled with an unrelated MoAb.
Statistical Analysis
Summary statistics, such as proportions, means,
standard deviations, medians, and ranges, were used to
describe the patient characteristics, pretransplant
variables, and posttransplant outcomes. All continuous
variables were compared using the Mann-Whitney
U-test. The differences in frequencies of the 2 groups
were compared using the chi-square test. All statistical
procedures were performed using SPSS, version 16.0.
AP\.05 was considered to be statistically significant.
RESULTS
Patient Characteristics
The demographic and clinical characteristics of the
allo-HSCT recipients with and without prolonged
thrombocytopenia are summarized in Table 1. All char-
acteristics except the transplanted CD34
1
cell dose,
platelet engraftment time, and history of CMV reactiva-
tion were nearly equally represented in the study and
control groups. As shown in Table 1, the observed differ-
ences in platelet engraftment time for the 2 groups were
significant (P5.002), and allo-HSCT patients with
276 Biol Blood Marrow Transplant 17:274-280, 2011X. Zhang et al.

prolonged thrombocytopenia exhibited slower platelet
engraftment times. We also found that allo-HSCT recip-
ients with prolonged thrombocytopenia had fewer trans-
planted CD34
1
cells (P\.001) in comparison to those
without. Additionally, more allo-HSCT recipients with
prolonged thrombocytopenia had undergone CMV re-
activation from the day when the patients accepted the
donors’ stem cells to the day of the bone marrow sam-
pling (P5.002); however, the recent CMV reactivation
and the recent use of ganciclovir at the time of bone mar-
row sampling were not significantly different between the
allo-HSCT recipients with prolonged thrombocytope-
nia and those without. There were 6 prolonged thrombo-
cytopenic patients and 1 nonthrombocytopenic patient
who had undergone current CMV reactivation when
the bone marrow tests were performed. For treatment,
3 of the 6 prolonged thrombocytopenic patients used
ganciclovir, 2 used foscarnet, and 1 used intravenous
gamma globulin. The nonthrombocytopenic patient
was treated with ganciclovir for the CMV infection.
The comparison of the current CMV reactivation and
antivirus treatment was not significantly different
between the prolonged thrombocytopenic and non-
thrombocytopenic allo-HSCT recipients. Additionally,
age and gender did not significantly differ between the
healthy volunteers and allo-HSCT recipients with or
without prolonged thrombocytopenia (P..05).
Characterization of the MK Population
The proportion of MKs and c-Mpl (CD110)
expression
There was no difference between the proportion of
MKs in the marrow of mononuclear cells in the allo-
HSCT patients with prolonged thrombocytopenia
(0.21% 60.19%), allo-HSCT patients without
prolonged thrombocytopenia (0.24% 60.25%), and
healthy volunteers (0.26% 60.26%) (P..05 for all
comparisons). The percentages of CD110
1
MKs were
not significantly different between the 3 groups (data
not shown).
MKs ploidy distribution
The analysis of the MK ploidy distribution in allo-
HSCT patients with prolonged thrombocytopenia
showed different patterns in comparison to those of
allo-HSCT recipients without prolonged thrombocy-
topenia or healthy volunteers, as shown in Figure 1.
Figure 1A depicts a quantitative analysis of the allo-
HSCT recipients with prolonged thrombocytopenia
MK ploidy. The MK ploidy peak was 2N and com-
prised 25.14% 613.20% of the total MKs, whereas
the proportions from 2N to 64N were frequently
decreased, which is a pattern that was not seen in
the patients without prolonged thrombocytopenia
Table 1. Clinical Characteristics of allo-HSCT Recipients with and without Prolonged Thrombocytopenia
Clinical Characteristics
Prolonged Thrombocytopenia
Present (n 532) Absent (n 527) PValue*
Age (year, median, range) 22 (8-51) 24 (6-52) 0.578
Male 23 (71.88%) 17 (62.96%) 0.579
Type of donor: related 32 (100.00%) 25 (92.59%) 0.205
Mismatched relatives 29 (90.63%) 19 (70.37%) 0.091
Stem-cell source
Bone marrow + peripheral blood 32 (100.00%) 25 (92.59%) 0.205
Peripheral blood 0 (0.00%) 2 (7.41%) 0.205
Transplanted stem-cell dose (10
8
/kg, median, range) 7.26 (4.31-9.93) 7.33 (4.25-11.55) 0.726
Transplanted CD34
+
cell dose (10
6
/kg, median, range) 1.51 (0.67-3.52) 2.40 (1.33-7.63) <0.001
Underlying disease
AML 14 (43.75%) 10 (37.04%) 0.791
ALL 6 (18.75%) 10 (37.04%) 0.147
MDS 2 (6.25%) 1 (3.70%) 1.000
NHL 1 (3.13%) 1 (3.70%) 1.000
CML 5 (15.63%) 3 (11.11%) 0.715
SAA 3 (9.38%) 2 (5.41%) 1.000
Aggressive NK cell leukemia 1 (3.13%) 0 (0.00%) 1.000
Platelet engraftment time (post-HSCT days) 17 (7-114)†12 (7-27) 0.002
WBC engraftment time (post-HSCT days) 13.5 (10-22) 12 (10-19) 0.594
History of GVHD 26 (81.25%) 18 (66.67%) 0.24
Current aGVHD‡2 (6.25%) 1 (3.70%) 1.000
Current extensive cGVHD 1 (3.13%) 1 (3.70%) 1.000
History of CMV reactivation 27 (84.38%) 12 (44.44%) 0.002
allo-HSCT indicates allogeneic hematopoietic stem cell transplantation; AML, acute myelogenous leukemia; ALL, acute lymphocytic leukemia; aGVHD,
acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease; MDS, myelodysplastic syndrome; NHL, non-Hodgkin lymphoma; CML,
chronic myelogenous leukemia; SAA, severe aplastic anemia; GVHD, graft-versus-host disease; CMV, cytomegalovirus; WBC, white blood cell.
*The continuous variables were compared using the Mann-Whitney U-test, and the differences in frequency between the 2 groups were compared using
the chi-square test.
†The data include all patients except for 3 who died before their platelets engrafted.
‡Grades II-IV.
Biol Blood Marrow Transplant 17:274-280, 2011 277MKs Ploidy and Platelet Recovery

(Figure 1B). We compared the ploidy distribution of
the MKs of allo-HSCT patients with prolonged
thrombocytopenia to those without. The former group
exhibited a marked increase in #8N cells (55.63% 6
18.62% versus 44.63% 619.38%, P5.036) and a sig-
nificant decrease in 16N and $32N cells (10.02 65.60
versus 17.72 67.23, P\.001; 9.18% 67.63% versus
20.28% 615.71%, P5.01, respectively). These re-
sults indicate that the allo-HSCT patients with pro-
longed thrombocytopenia exhibited a significant shift
to a population of low ploidy cells (left shift), suggest-
ing that there were more less-mature MKs in the pa-
tients of this group.
In contrast, the modal ploidy for the healthy volun-
teers was 16N, which comprised 37.12% 610.47% of
the total MKs; 25.97% 610.70% of the MKs had
a ploidy #8N, whereas 22.21% 64.81% of the total
MKs had a ploidy $32 N (Figure 1C). In both
allo-HSCTpatients with and without prolonged throm-
bocytopenia, the MK ploidy distribution exhibited
a significant shift to low ploidy cell populations (left
shift), particularly in the patients with prolonged throm-
bocytopenia. In comparison to healthy volunteers,
the prolonged thrombocytopenic patients exhibited
a marked increase primarily in #8N cells (55.63% 6
18.62%, P\.001) and a reciprocal decrease in both
16N (10.02% 65.60%, P\.001) and $32N cells
(9.18% 67.63%, P\.001). In the nonthrombocyto-
penic allo-HSCT recipients, the MK ploidy distribution
also shifted toward a low ploidy population (left shift),
wherein 44.63% 619.38% of cells had a ploidy of
#8N (P5.003, in comparison to healthy volunteers).
The percentage of 16N cells (17.72% 67.23%) in the
nonthrombocytopenic allo-HSCT recipients was still
less than that of healthy volunteers (P\.001); however,
the proportions of $32N cells in the group without
prolonged thrombocytopenia post-HSCT were compa-
rable to those of healthy volunteers (P5.081).
DISCUSSION
Few studies have examined the relationship be-
tween post-transplantation MK differentiation and
maturation and thrombocytopenia in allo-HSCT pa-
tients. In previous studies, bone marrow smears or biop-
sies were used to count bone marrow MKs;however, the
obtaining of bone marrow specimens is difficult and re-
quires the technical skills of an experienced healthcare
professional. The study of human MKs in vivo has
proven to be difficult because of their relative rarity, fra-
gility, and inherent tendency to aggregate [17,18].
Consequently, highly selective methods are needed
for the successful isolation and analysis of human
MKs. Flow cytometry offers several advantages in the
study of the DNA content of MKs. This technique
enables the rapid identification of large numbers of
MKs, and their ploidy number can be reliably
determined [15].
Prolonged thrombocytopenia after allo-HSCT
may result from complex mechanisms; however, no
previous studies have clearly described the characteris-
tics of marrow MKs, particularly in regard to their
ploidy distribution, which is associated with MK mat-
uration. In the present study, we found that platelet
engraftment was slower in allo-HSCT recipients
with prolonged thrombocytopenia in comparison to
that in nonthrombocytopenic allo-HSCT recipients.
Figure 1. The megakaryocyte ploidy distribution pattern of the 3
groups (shown as the means of the relative frequency and standard de-
viation). (A) The allo-HSCT patients with prolonged thrombocytopenia
(n 532); (B) The allo-HSCT patients without prolonged thrombocyto-
penia (n 527). (C) The group of healthy volunteers (n 513). The modal
megakaryocyte of healthy volunteers ploidy is 16N. The megakaryocyte
ploidy distribution in the allo-HSCT patients, especially in the patients
with prolonged thrombocytopenia, exhibited a significant shift to low
ploidy cells (left shift), with a marked increase in 2N to 8N cells and
a reciprocal decrease in 16N to 64N cells.
278 Biol Blood Marrow Transplant 17:274-280, 2011X. Zhang et al.

Using flow cytometry, the cause of the prolonged
thrombocytopenia and slow platelet engraftment in
allo-HSCT recipients were evaluated by measuring
the percentages and ploidy distributions of marrow
MKs. The comparable proportions of MKs and
c-Mpl expression in MKs between allo-HSCT recipi-
ents and healthy controls indicate that the slower
platelet engraftment and prolonged thrombocytope-
nia after allo-HSCT was not associated with the 2 vari-
ables, and therefore, that other causes must contribute
to prolonged thrombocytopenia.
The ploidy distribution pattern of the normal con-
trols (modal ploidy 5l6N) is consistent with previous
reports [15,16]; however, analysis of the ploidy
distribution pattern in allo-HSCT recipients with or
without prolonged thrombocytopenia exhibited a shift
toward a low ploidy cell population (left shift). In con-
trast, the proportions of MKs with ploidy classes of
2N to 8N were more predominant in allo-HSCT pa-
tients withprolonged thrombocytopenia in comparison
to patients without prolonged thrombocytopenia. The
above results may reflect a deregulation in the matura-
tion process in allo-HSCT patients, particularly in
those with prolonged thrombocytopenia. Because the
number of platelets that are generated from each MK
positively correlates with its DNA content and ploidy,
a reduced platelet number will occur if most MKs
have a lower ploidy content [19]. The slow platelet
engraftment and prolonged thrombocytopenia post
allo-HSCT may be because of the left shift of marrow
MK ploidy. Mature MKs increase their polyploidy by
a process known as ‘‘endomitosis,’’ which is a modified
cell cycle process in which several rounds of DNA rep-
lication occur without cytokinesis [20,21].Theincrease
in DNA content is associated with the development of
multilobated nuclei and increases in cytoplasmic
volume and cell surface area. This is followed by the
extension of proplatelets (long-branched cytoplasmic
protrusions) from which platelets are released [22,23].
The polyploidization process is not necessary to reach
a full MK differentiation but is necessary to release
ahighnumberofplatelets[24]. A shift in ploidy to
the left may reflect selective damage to the more mature
MKs or interference with the maturation process. In ad-
dition, MK hypoplasia may be present in allo-HSCT
recipients, particularly those with prolonged thrombo-
cytopenia; however, the mechanism for this shift is un-
known. The marrow MKs in allo-HSCT patients may
need a period of recovery after allo-HSCT, which is
a hypothesis that will need to be further investigated.
The causes of the left shift in the MK ploidy distri-
bution and the late recoveries of platelet counts in allo-
HSCT recipients with prolonged thrombocytopenia
remain obscure. Previous studies have reported that
megakaryocyte ploidy and platelet count fall during
the myelosuppression from chemotherapy [25],which
suggests that myelosuppression from conditioning ther-
apy for allo-HSCT recipients may play a predominant
role in the left shift of megakaryocyte ploidy; however,
the conditioning therapy for the allo-HSCT recipients
with prolonged thrombocytopenia did not differ from
that of the allo-HSCT recipients without prolonged
thrombocytopenia. We believe that other unknown
causes may be responsible, and these causes need to be
further characterized.
In the present study, we found that although the
number of transplanted stem cells was equal, the number
of CD34
1
cells was significantly lower in allo-HSCT
recipients with prolonged thrombocytopenia. Both
transplanted CD34
1
cell dose and cytomegalovirus in-
fection have been reported as important factors that cor-
relate with platelet recovery after hematopoietic stem
cell transplantation. A larger transplanted CD34
1
cell
count has been positively correlated with rapid platelet
engraftment [26,27].Therefore,alowernumberof
transplanted CD34
1
cells may affect the maturation
process of marrow MKs in allo-HSCT patients with
prolonged thrombocytopenia and lead to an increase
in the number of lower-ploidy MKs; however, this sce-
nario needs to be furtherexamined. In addition, a higher
frequency of allo-HSCT recipients with prolonged
thrombocytopenia had a history of CMV reactivation,
whereas at the time of bone marrow sampling, the 2
groups had comparable proportions of CMV reactiva-
tion and ganciclovir usage. Cytomegalovirus infection
can delay platelet recovery after hematopoietic stem
cell transplantation [28], and ganciclovir may also be as-
sociated with thrombocytopenia because of a decreased
production in platelets. We suspect that both of these
factors may delay megakaryocyte maturation; however,
it would be difficult to separate the effects of CMV or
ganciclovir on megakaryocytes.
In conclusion, prolonged thrombocytopenia and
slow platelet engraftment after allo-HSCT may be re-
lated to a reduction in ploidy and the immaturation of
MKs. Therefore, promotion of the MK maturation
process should be considered as a strategy for the treat-
ment of thrombocytopenia following allo-HSCT.
ACKNOWLEDGMENTS
This work was supported by the National Science
Foundation of China (Grant no. 30770911) and the
Program for Innovative Research Team in University
(Grant no. IRT 0702). The authors declare no con-
flicts of interest.
Financial disclosure: The authors have nothing to
disclose.
REFERENCES
1. Kim DH, Sohn SK, Jeon SB, et al. Prognostic significance of
platelet recovery pattern after allogeneic HLA-identical sibling
Biol Blood Marrow Transplant 17:274-280, 2011 279MKs Ploidy and Platelet Recovery

transplantation and its association with severe acute GVHD.
Bone Marrow Transplant. 2006;37:101-108.
2. Kim DH, Sohn SK, Baek JH, et al. Clinical significance of plate-
let count at day 160 after allogeneic peripheral blood stem cell
transplantation. J Korean Med Sci. 2006;21:46-51.
3. Bolwell B, Pohlman B, Sobecks R, et al. Prognostic importance
of the platelet count 100 days post allogeneic bone marrow
transplant. Bone Marrow Transplant. 2004;33:419-423.
4. First LR, Smith BR, Lipton J, Nathan DG, Parkman R,
Rappeport JM. Isolated thrombocytopenia after allogeneic
bone marrow transplantation: existence of transient and chronic
thrombocytopenic syndromes. Blood. 1985;65:368-374.
5. Bielski M, Yomtovian R, Lazarus HM, Rosenthal N. Prolonged
isolated thrombocytopenia after hematopoietic stem cell trans-
plantation: morphologic correlation. Bone Marrow Transplant.
1998;22:1071-1076.
6. Yamazaki R, Kuwana M, Mori T, et al. Prolonged thrombocyto-
penia after allogeneic hematopoietic stem cell transplantation:
associations with impaired platelet production and increased
platelet turnover. Bone Marrow Transplant. 2006;38:377-384.
7. Ebbe S. Biology of megakaryocytes. Prog Hemost Thromb. 1976;
3:211-229.
8. Gewirtz AM. Megakaryocytopoiesis: the state of the art. Thromb
Haemost. 1995;74:204-209.
9. Hoffman R. Regulation of megakaryocytopoiesis. Blood. 1989;
74:1196-1212.
10. Huang XJ, Liu DH, Liu KY, et al. Haploidentical hematopoietic
stem cell transplantation without in vitro T-cell depletion for
the treatment of hematological malignancies. Bone Marrow
Transplant. 2006;38:291-297.
11. Lu DP, Dong L, Wu T, et al. Conditioningincluding antithymo-
cyte globulin followed by unmanipulated HLA-mismatched/
haploidentical blood and marrow transplantation can achieve
comparable outcomes with HLA-identical sibling transplanta-
tion. Blood. 2006;107:3065-3073.
12. Shulman HM, Sullivan KM, Weiden PL, et al. Chronic graft-
versus-host syndrome in man. A long-term clinicopathologic
study of 20 Seattle patients. Am J Med. 1980;69:204-217.
13. Thomas ED, Storb R, Clift RA, et al. Bone-marrow transplan-
tation (second of two parts). N Engl J Med. 1975;292:895-902.
14. Tomer A, Friese P, Conklin R, et al. Flow cytometric analysis of
megakaryocytes from patients with abnormal platelet counts.
Blood. 1989;74:594-601.
15. Tomer A. Human marrow megakaryocyte differentiation: mul-
tiparameter correlative analysis identifies von Willebrand factor
as a sensitive and distinctive marker for early (2N and 4N) mega-
karyocytes. Blood. 2004;104:2722-2727.
16. Tomer A, Harker LA. Measurements of in vivo megakaryocyto-
poiesis: studies in nonhuman primates and patients. Stem Cells.
1996;14(Suppl 1):18-30.
17. Levine RF. Isolation and characterization of normal human
megakaryocytes. Br J Haematol. 1980;45:487-497.
18. Nakeff A. Megakaryocytic cells. Bibl Haematol. 1984;131-209.
19. Harker LA, Finch CA. Thrombokinetics in man. J Clin Invest.
1969;48:963-974.
20. Vitrat N, Cohen-Solal K, Pique C, et al. Endomitosis of human
megakaryocytes are due to abortive mitosis. Blood. 1998;91:
3711-3723.
21. Zimmet J, Ravid K. Polyploidy: occurrence in nature, mecha-
nisms, and significance for the megakaryocyte-platelet system.
Exp Hematol. 2000;28:3-16.
22. Italiano JE Jr., Lecine P, Shivdasani RA, Hartwig JH. Blood
platelets are assembled principally at the ends of proplatelet pro-
cesses produced by differentiated megakaryocytes. J Cell Biol.
1999;147:1299-1312.
23. Patel SR, Richardson JL, Schulze H, et al. Differential roles of
microtubule assembly and sliding in proplatelet formation by
megakaryocytes. Blood. 2005;106:4076-4085.
24. Mattia G, Vulcano F, Milazzo L, et al. Different ploidy levels of
megakaryocytes generated from peripheral or cord blood
CD341cells are correlated with different levels of platelet
release. Blood. 2002;99:888-897.
25. Bessman D. Prediction of platelet production during che-
motherapy of acute leukemia. Am J Hematol. 1982;13:
219-227.
26. Keever-Taylor CA, Klein JP, Eastwood D, et al. Factors affect-
ing neutrophil and platelet reconstitution following T cell-
depleted bone marrow transplantation: differential effects of
growth factor type and role of CD34(1) cell dose. Bone Marrow
Transplant. 2001;27:791-800.
27. Singhal S, Powles R, Treleaven J, et al. A low CD341cell dose
results in higher mortality and poorer survival after blood or
marrow stem cell transplantation from HLA-identical
siblings: should 2 10(6) CD341cells/kg be considered the
minimum threshold? Bone Marrow Transplant. 2000;26:
489-496.
28. Verdonck LF, de Gast GC, van Heugten HG, Nieuwenhuis HK,
Dekker AW. Cytomegalovirus infection causes delayed platelet
recovery after bone marrow transplantation. Blood. 1991;78:
844-848.
280 Biol Blood Marrow Transplant 17:274-280, 2011X. Zhang et al.