Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3286-3293
The Effect of Recombinant Human Erythropoietin on Platelet Counts Is
Strongly Modulated by the Adequacy of Iron Supply
By
Martine Loo and
Yves Beguin
From the Department of Medicine, Division of Hematology, University
of Liège, Liège, Belgium.
 |
ABSTRACT |
The effect of recombinant human erythropoietin (rHuEpo) on
megakaryopoiesis remains controversial. Treatment with rHuEpo in renal
failure patients has been associated with a slight elevation of
platelet counts. In animal studies, high doses of rHuEpo produced an
increase of platelet counts followed by a gradual return to normal
after 7 to 15 days or even a substantial degree of thrombocytopenia. However, because iron deficiency is also known to be associated with
thrombocytosis, (functional) iron deficiency during rHuEpo could be
contributing to these observations. We investigated the impact of iron
supply on changes in platelet counts induced by rHuEpo. Rats were
either fed normal food (normal rats) or received 1% carbonyl iron for
2 weeks or 3 months, as well as during the experiment, to achieve iron
supplementation or overload, respectively. Rats of all three categories
then received daily intravenous injections of rHuEpo (10, 50, or 150 U)
or normal saline (0 U) for 20 days. With 0 to 10 U rHuEpo, platelets
remained stable. In normal rats receiving 50 to 150 U rHuEpo, platelets
increased to 120% to 140% of baseline at 4 to 12 days to level off at
120% at 16 to 20 days. This response was less sustained in
splenectomized animals. Iron-supplemented rats receiving 50 to 150 U
rHuEpo also increased platelets initially, but the peak was at day 4, followed by a gradual return to baseline and even a moderate
thrombocytopenia later on. Iron-overloaded rats receiving 50 to 150 U
rHuEpo also had increased platelets at day 4, but the duration of
platelet increase was shorter, and they experienced a more pronounced
degree of thrombocytopenia in proportion to the dose of rHuEpo. Because
the early elevation of platelets was of larger magnitude than
hematocrit changes, it is unlikely that it could be
accounted for by shrinkage of plasma volume. Because it was observed in
all three iron conditions, there appears to be some direct positive
effect of rHuEpo on platelet production. However, after this transient
effect, expanded erythropoiesis appears to exert a negative impact upon
platelet production. Secondary thrombocytopenia was not related to
splenic pooling, and its very slow correction after cessation of rHuEpo
therapy is not compatible with changes in platelet survival. Rather, it
is consistent with stem cell competition between erythroid and
megakaryocytic development. However, this secondary thrombocytopenia is
masked by (functional) iron deficiency in rats not receiving an
adequate iron supply from food or stores.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ERYTHROPOIETIN (Epo) is the primary
erythropoietic growth factor, but it has also been shown to affect
platelet production. In large clinical trials of recombinant human Epo
(rHuEpo) in renal failure patients, platelet counts increased
significantly within 2 weeks and then gradually returned to
pretreatment levels after months of maintenance therapy.1,2
Mean platelet volumes decreased, but platelet counts remained unchanged
in patients receiving rHuEpo and oral iron before scheduled
surgery.3 In adult but not in infant monkeys, rHuEpo
therapy resulted in elevated platelet counts throughout the 6-week
treatment, followed by rapid normalization thereafter.4 In
dogs, mice, or rats, short-term treatment with high doses of rHuEpo has
been shown to stimulate platelet production, but platelet counts tended
to normalize after 7 to 15 days.5-9 However, transgenic
mice expressing the human Epo gene develop both polycythemia and a
moderate degree of thrombocytopenia.10 Actually, large,
chronic doses of rHuEpo also caused thrombocytopenia in mice, and stem
cell competition between erythroid and platelet precursors has been
suggested as the cause of this phenomenon.11
In a previous study of patients with end-stage renal disease, we
demonstrated that platelet changes in response to rHuEpo therapy
correlated with modifications of erythropoietic activity.2 However, enhanced erythropoiesis is also associated with modifications of iron metabolism. In particular, functional iron deficiency, ie, an
imbalance between iron needs in the bone marrow and iron supply from
stores, may develop even in the presence of adequate storage iron when
these stores cannot be mobilized rapidly enough.12 Iron
deficiency has been shown to be associated with reactive thrombocytosis.13-17 Iron supplementation in iron-deficient
infants18 or rats19,20 was rapidly followed by
a return of platelet counts to normal levels.
Therefore, we undertook the present study to investigate the impact of
iron supply on changes in platelet counts induced by rHuEpo. We studied
the effect of various doses of rHuEpo and various durations of
treatment on both erythropoiesis and megakaryopoiesis. We also examined
the potential role of the spleen in this setting, because some studies
had suggested that splenic pooling could interfere with these
observations.7,21,22 We observed a diphasic pattern of
response of platelet counts to rHuEpo, with the initial increment of
platelet counts being followed by substantial thrombocytopenia. The
study demonstrated that the iron status of the animals played an
important role in the second phase of the diphasic response, with iron
deficiency protecting them from thrombocytopenia.
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MATERIALS AND METHODS |
Animals.
Male Wistar rats were obtained from Janssen (Beerse, Belgium) and
raised at the University of Liège (Liège, Belgium) from the
age of 3 weeks through the end of the experiment. One group of animals
(normal rats) was fed standard granular food. Other groups of rats were
fed standard food in powder form supplemented with 1% (wt/wt) carbonyl
iron (Sigma Chemical Co, St Louis, MO) 6 days per week and normal
granular food 1 day per week, for a total of 2 weeks (supplemented
rats) or 3 months (overloaded rats) before as well as during the
experiment, to achieve iron supplementation or iron overload,
respectively. Some animals underwent splenectomy under general
anesthesia with ether (Gifer Barbezat, Dicenes, France) followed by
placement of Autoclip Mikron clips (Becton Dickinson Benelux,
Erembodegem-Alst, Belgium) for 10 days. They were then left to
stabilize for 30 days before the experiment, and only animals showing
no sign of inflammation at that time were used. Other rats underwent
the same procedure but without splenectomy (sham-operated).
Epo therapy.
rHuEpo (Recormon) was kindly provided by Boehringer Mannheim (Brussels,
Belgium). Rats were injected daily with intravenous rHuEpo at a dose of
10, 50, or 150 U/d for up to 20 days. Control animals (0 U rHuEpo)
received the same volume of normal saline.
Laboratory analyses.
Blood samples were drawn from a tail vein under short ether anesthesia
3 times per week just before rHuEpo injections. Part of the blood was
drawn on ACD formula A and part on heparin to be centrifuged to obtain
plasma, which was frozen at 
20°C until processing. Complete
blood counts were measured on a Technicon H1 automatic cell counter
(Technicon, Tarrytown, NY), with appropriate corrections for dilution
by ACD. Preliminary experiments showed that a minor proportion of the
smaller rat platelets could not be detected by the counter. However,
this fraction was constant from rat to rat as well as over time and
platelet volume did not change during rHuEpo therapy. Expressing the
results as the percentage of baseline values thus eliminated this
problem. The small fraction of larger platelets was appropriately
detected. The percentage of reticulocytes was determined by
cytofluorometry on a FACSCAN cytofluorometer (Becton Dickinson, San
Jose, CA) after coloration with thiazole orange.23
Preliminary experiments showed that this automated method gave results
similar to manual counting on blood smears colored with brilliant
cresyl blue. Plasma soluble transferrin receptor (sTfr) was measured as
previously described with minor modifications.24,25 After
varying durations of treatment, a subset of the animals were
exsanguinated from the abdominal aorta and perfused with 20 to 30 mL
saline solution. The spleen was removed and weighted. Nonheme iron was
extracted from the liver,26 and iron concentration was
determined by proton-induced x-ray emission.27 Serum iron
(SeFe) and total iron binding capacity (TIBC) were measured using
standard methods,28 and transferrin saturation was derived
from these figures.
Statistical methods.
All results were expressed as percentages of a baseline value and
expressed as the mean ± standard deviation (M ± SD).
Comparisons of baseline values with later measurements in the same
group of animals were performed using paired Student's
t-tests. Comparisons between groups were performed with
Student's t-tests, with Welsch's correction in case of
unequal variances. Most statistical analyses were performed with the
Excel 97 (Microsoft Corp, Redmond, WA) or Prism 2.0 (GraphPad Software
Inc, San Diego, CA) software.
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RESULTS |
During treatment with rHuEpo, a progressive erythroid response was
observed and its amplitude was similar in unmanipulated, sham-operated,
or splenectomized animals. This response was proportional to the dose
of rHuEpo (Figs 1 and
2). Low doses (10 U) of rHuEpo produced
some increment of hematocrit (Hct; Fig 1) and
reticulocytes (Fig 2) without any change in sTfR levels, suggesting
that this was achieved through shift reticulocytosis rather than true
stimulation of erythropoiesis. Higher doses caused more substantial
increases of sTfR, reticulocytes, and Hct. The iron status of the
animals strongly modulated the erythroid response, being significantly more prominent in overloaded rats compared with supplemented and normal
rats, even if the initial slope of Hct increase was similar in
overloaded compared with normal rats (Fig
3). After 20 days of treatment with 150 U rHuEpo, the Hct of overloaded
rats attained a plateau at 160% of baseline values, whereas normal
rats only achieved a 135% plateau (Fig 1). Reticulocytes attained
levels 7 times versus 3 times baseline in overloaded and normal
animals, respectively (Fig 2).
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| Fig 1.
Evolution of Hct during treatment with daily doses of 0 (diamonds), 10 (triangles), 50 (circles), or 150 (squares) U rHuEpo.
Results are expressed as percentages of baseline value. (A) Normal rats
(solid symbols); (B) overloaded rats (open symbols).
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| Fig 2.
Evolution of absolute reticulocyte count during treatment
with daily doses of 0 (diamonds), 10 (triangles), 50 (circles), or 150 (squares) U rHuEpo. Results are expressed as percentages of baseline
value. (A) Normal rats (solid symbols); (B) overloaded rats (open
symbols).
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| Fig 3.
Evolution of Hct, absolute reticulocyte count,
and sTfR during treatment with daily doses of 50 U rHuEpo in normal
( ) or overloaded ( ) rats. Results are expressed as percentages of
baseline value. (A) Hct; (B) absolute reticulocyte count; (C) sTfR.
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Spleen weight remained stable with 0 or 10 U rHuEpo, but increased
significantly in all categories of animals receiving 50 to 150 U per
day (Table 1). The storage (liver) and
functional (plasma) iron pools were not modified with 0 or 10 U rHuEpo,
but when modified with higher doses of rHuEpo, were significantly depleted compared with baseline (Table 1). With 50 to 150 U rHuEpo, normal rats showed depletion of the functional as well as storage pools
consistent with iron deficiency, supplemented rats had functional iron
deficiency but iron stores were equivalent to those of untreated normal
rats, and overloaded rats retained adequate plasma iron levels in the
presence of elevated stores.
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Table 1.
Serum Iron, Liver Nonheme Iron, and Spleen Weight in
Normal, Supplemented, and Overloaded Rats Treated for 0, 4, or 16 Days With 0, 10, 50, or 150 U rHuEpo Per Day
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In normal rats, treatment with rHuEpo produced dose-dependent effects
on platelet counts (Fig 4A). With 10 U
rHuEpo per day, platelets remained stable and comparable to levels
observed with 0 U. On the other hand, with 50 U rHuEpo platelet counts
increased to 120% of baseline after 4 days and remained stable
thereafter. With 150 U rHuEpo daily, they increased to 140% of
baseline by day 8 to progressively level off at 120% at 14 to 20 days
(Figs 4A and 5). In animals receiving 0 U
rHuEpo, supplemented or overloaded (Fig 4B) rats behave similarly to
normal rats. Supplemented rats receiving 50 to 150 U rHuEpo also
increased platelets initially, but the peak was at day 4, followed by a
gradual return to baseline by day 10 and a moderate degree of
thrombocytopenia later on (Fig 5). Overloaded rats receiving
50 to 150 U rHuEpo also increased platelets at day 4. However, the
duration of platelet increase was shorter and the importance of
secondary thrombocytopenia was more pronounced (Figs 4B and 5). This
occurred in proportion to the dose of rHuEpo and reached 60% of
baseline with 150 U rHuEpo per day for 20 days (Fig 4B).
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| Fig 4.
Evolution of platelet counts during treatment with daily
doses of 0 (diamonds), 10 (triangles), 50 (circles), or 150 (squares)
U rHuEpo. Results are expressed as percentages of baseline
value. (A) Normal rats (solid symbols); (B) overloaded rats (open
symbols).
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| Fig 5.
Evolution of platelet counts during treatment with daily
doses of 150 U rHuEpo in normal ( ), supplemented ( ), or
overloaded ( ) rats. Results are expressed as percentages of baseline
value.
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In normal animals previously splenectomized, treatment with high doses
of rHuEpo was associated with a transient elevation of platelet counts
peaking at day 4 before progressively returning to baseline, whereas
unmanipulated animals maintained increased platelet counts throughout
the experiment (Fig 6A). This
contrasted with supplemented or overloaded rats (Fig 6B), in
which unmanipulated, sham-operated, or splenectomized animals behave
similarly, ie, first increased platelet counts and then developed
secondary thrombocytopenia with high doses of rHuEpo.
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| Fig 6.
Evolution of platelet counts during treatment with daily
doses of 150 U rHuEpo in unmanipulated (solid symbols), sham-operated
(shaded symbols), or splenectomized (open symbols) rats. Results are
expressed as percentages of baseline value. (A) Normal rats (diamonds);
(B) overloaded rats (squares).
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Treatment with 150 U rHuEpo was stopped after 20 days and rats were
followed-up for 30 days after cessation of therapy
(Fig 7). The Hct, reticulocytes,
and sTfR levels decreased progressively, whereas platelet counts
recovered in parallel over a period of 4 weeks. The evolution was
identical in unmanipulated, sham-operated, or splenectomized animals.
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| Fig 7.
Evolution of Hct (---) and platelet counts
( ) after cessation of treatment with daily doses of 150 U rHuEpo in
overloaded rats. Results are expressed as percentages of baseline
value.
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DISCUSSION |
In the present study, animals receiving 10 U rHuEpo did not experience
changes in platelet counts but had some increase in Hct and
reticulocytes. However, as this happened without mobilization of
storage iron and without increase in sTfR levels (the best quantitative
measure of total erythropoiesis), this was probably achieved through
shift reticulocytosis rather than true stimulation of erythropoietic
activity. Rats treated with higher doses of rHuEpo underwent a true
expansion of erythropoiesis, translating into progressive elevation of
all erythroid parameters. Normal animals soon became limited in their
capacity to respond because of the occurrence of iron deficiency. The
augmentation of erythropoietic activity continued further in overloaded
rats and these animals attained much higher sTfR values. Soluble TfR
levels, thought in some situations to be a marker of (functional) iron
deficiency,24 are therefore much more sensitive to changes
in total erythropoietic activity than to changes in iron availability.
This is consistent with our previous finding that patients with
functional iron deficiency before treatment did not increase sTfR
levels in response to rHuEpo.29
Our results confirm that high doses of rHuEpo induce significant
changes in platelet counts. The platelet response appeared to undergo
two different phases, ie, an initial moderate thrombocytosis and a
secondary more pronounced thrombocytopenia. This diphasic pattern of
response has already been described in animal models of
hypoxia30-33 but not in models of rHuEpo therapy.
The initial increment in platelet counts was initiated within 48 hours
of starting rHuEpo therapy and developed in a dose-dependent fashion.
This short-term elevation of platelet counts could be accounted for by
several mechanisms, including changes in plasma volume or platelet
survival and stimulation of megakaryocyte activity or global marrow
function. Proportional shrinkage of plasma volume occurs only after
rHuEpo treatment has produced changes in the red blood cell (RBC)
mass.34 Furthermore, simultaneous elevations of RBCs and
platelets should then be observed, but this was not the case in renal
failure patients.2 An effect of rHuEpo on platelet survival
has never been demonstrated, and there is no reason why this would be
limited to patients in whom RBC production is adequately
stimulated.2 A number of in vitro data support the concept
that Epo directly stimulates megakaryopoiesis. Megakaryocytes have been
shown to express constitutive35 or
inducible36,37 high-affinity binding sites for Epo,
resulting in enhanced growth in the presence of Epo.38 In
vitro studies in humans as well as in mice have demonstrated that Epo
promotes megakaryocytic colony formation and increases the size,
ploidy, and number of megakaryocytes, as well as their DNA and protein
synthesis and cytoplasmic process formation.38-42 Instead
of directly stimulating megakaryocytes, Epo could also promote platelet
production indirectly by enhancing global hematopoietic activity.
Treatment of patients with end-stage renal failure rapidly resulted in
an increase of not only erythroid progenitors, but also of
colony-forming units megakaryocytes (CFU-Meg) and colony-forming
unit-granulocyte-macrophage (CFU-GM).43 In
such patients, platelet increments correlated with the degree of
expansion of erythropoietic activity and platelet counts did not change
in nonresponders until the Epo dose was increased and erythropoiesis
begun to expand.2 In chronic liver disease, rHuEpo therapy
ameliorates platelet counts in patients who also show a response of the
erythroid lineage.44 In mice, elevated platelet counts
correlated with increased Hct after 5 days of rHuEpo
therapy.7 These observations suggest that the Epo effect on
platelet formation parallels its effect on RBC production.
In animals with adequate iron supply, the initial elevation of platelet
counts rapidly gave way to a substantial degree of thrombocytopenia.
This also developed in a dose-dependent manner, with the decrease
occurring earlier and being more pronounced in animals receiving higher
doses of rHuEpo. In previous studies with rHuEpo, the initial elevation
of platelet counts was followed by a return of platelets to control
levels after 7 or 15 days.6,7 Actually, large, chronic
doses of rHuEpo caused thrombocytopenia, decreased seleno-methionine
incorporation into platelets, and reduced number of megakaryocytes in
normal mice.11 Transgenic mice expressing the human Epo
gene developed both polycythemia and a moderate degree of
thrombocytopenia.10 Similarly, chronic hypoxia decreased
platelet production,30-33,45 and this was shown to result
from decreased differentiation of hematopoietic precursors into the
megakaryocytic lineage.31,46 Increased erythropoiesis and
not elevated RBCs (as produced by transfusion) was required for the
thrombocytopenia to occur.33,47 Conversely, acute
thrombocytopenia caused decreased erythropoiesis in some
studies.11 Short-term thrombopoietin (Tpo)
treatment in normal mice48 as well as chronic exposure to
Tpo after gene transfer49 induced erythroid hypoplasia. However, this was at least partially explained by the development of
myelofibrosis. Finally, Epo-induced thrombocytopenia is very unlikely
to be due to increased platelet destruction, as observed with other
growth factors such as macrophage colony-stimulating factor
(M-CSF).50 During Epo-induced
thrombocytopenia, there is substantial suppression of
megakaryopoiesis11,31,46 and no change in platelet
survival.30 Furthermore, recovery of a normal platelet
count took several weeks after cessation of rHuEpo therapy, a finding
difficult to reconcile with recovery after ending activation of the
reticulo-endothelial system.
All this is suggestive of a pattern of reciprocal changes in
erythropoiesis and megakaryopoiesis. Therefore, stem cell competition between erythroid and platelet precursors has been suggested to occur
in these situations of prolonged, intense stimulation.11 Recent data support the concept that megakaryocytic and erythrocytic cell lineages share a common precursor.51
Multipotential37 or bipotential52-54 cell lines
expressing Epo receptors37,53 have been obtained in which
erythroid differentiation can be induced through the action of
Epo37,52 and megakaryocytic differentiation by
Tpo.52,54 Such a bipotent erythro-megakaryocytic progenitor has also been characterized in normal human bone marrow,55
coexpressing glycophorin A and glycoprotein IIIa.56
The effect of rHuEpo on platelet counts was strongly modulated by the
iron status of the animals. Thrombocytopenia occurred earlier and was
more pronounced in overloaded rats (no iron deficiency) than in
supplemented animals (functional iron deficiency). Normal animals
developed iron deficiency with depletion of iron stores that resulted
in apparent protection from Epo-induced thrombocytopenia. Iron
deficiency has been shown to be associated with
thrombocytosis,13-17 but platelet counts tend to
normalize19,20,57 and thrombocytopenia can even
occur18,58,59 when iron deficiency becomes very severe. This is consistent with the diphasic pattern of majored stimulation by
endogenous Epo. Iron therapy ensures correction of platelet counts in
infants18 or in rats19,20 with moderate iron
deficiency, but has little effect in rats with severe iron deficiency
anemia and normal platelet counts.19,20 In cases with very
severe iron deficiency, iron therapy may even be associated with
thrombocytopenia.60 Therefore, the effects of iron status
and iron therapy on platelet production depend on the severity of iron deficiency.
However, changes in platelet counts during iron deficiency anemia could
represent in part an effect of increased endogenous Epo stimulation in
response to the anemia rather than an effect of iron deficiency per se.
With moderate iron deficiency, a modest increase in serum Epo would
stimulate platelet production, whereas with more severe anemia a major
Epo response would engender thrombocytopenia. Accordingly, transfusion
of RBCs into iron-deficient rats decreases platelet production, whereas
transfusion into normal animals has no effect.20 In
patients with renal failure receiving moderate doses of rHuEpo,
relative platelet increments over baseline correlated inversely with
relative changes of serum iron or transferrin saturation (an indication
of erythroid marrow activity) rather than with absolute serum iron and
transferrin saturation values (an indication of functional iron
deficiency) or with ferritin levels (an indication of iron stores),
emphasizing the role of marrow response to rHuEpo rather than that of
iron deficiency alone.2 Our results in overloaded animals
also show that the early stimulatory effect of rHuEpo on platelet
production is a direct effect of treatment rather than an indirect
effect of iron deficiency. Nevertheless, our data also show that with
high doses of rHuEpo thrombocytopenia develops in inverse relationship
to the degree of iron deficiency. This indicates that iron deficiency
per se plays an important protective role against thrombocytopenia
induced by high doses of rHuEpo in the presence of adequate iron supply.
Normal splenectomized animals showed a much less sustained platelet
response to rHuEpo therapy compared with unmanipulated rats, but this
discrepancy was no longer apparent in supplemented or overloaded rats
(Fig 6). Previous studies in mice have shown that the stimulation
effect of rHuEpo on megakaryocyte and CFU-Meg numbers may be limited to
the spleen in intact mice and become significant in the bone marrow
only after splenectomy.7,21,22 Splenectomized animals may
thus be unable to maintain the positive response to rHuEpo observed in
intact animals. It could also be possible that these rats experienced a
higher degree of marrow erythroid expansion compensating for the
absence of the spleen, thereby creating more competition with platelet
production. However, this was no longer true in supplemented or
overloaded rats. Splenic hematopoiesis may be less sensitive to
Epo-induced competition, explaining why hypoxia is associated with a
normal absolute number of megakaryocytes in the spleen61,62
and why splenectomy causes a further degree of hypoxia-induced
thrombocytopenia in hypertransfused mice.63
A question remaining to be examined in further studies is whether the
effects of rHuEpo on platelet production are a result of the dose
itself or of the magnitude of the erythropoietic effect of that dose.
For example, could a lower dose administered in a patient with
decreased marrow function (because his number of hematopoietic
progenitors is decreased by postchemotherapy stem cell damage or in the
context of transplantation) bring about the same biological effects as
those induced by higher doses of rHuEpo in the presence of a normal
marrow function?
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FOOTNOTES |
Submitted October 30, 1998; accepted December 9, 1998.
Supported in part by Grants No. 3.4555.91, 3.4621.94, and 7.4538.96 from the FNRS. M.L. was supported by a grant from
"Télévie" (FNRS, Belgium) and by a grant from the
"Fondation Frédéricq" (University of Liège,
Belgium). Y.B. is a Senior Research Associate of the National Fund for
Scientific Research (FNRS, Belgium).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Yves Beguin, MD, University of Liège,
Department of Hematology, CHU Sart-Tilman, 4000 Liège, Belgium;
e-mail: yves.beguin{at}chu.ulg.ac.be.
| |
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TOP
ABSTRACT
INTRODUCTION
