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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3451-3456
Elevated Levels of Circulating Procoagulant Microparticles in
Patients With Paroxysmal Nocturnal Hemoglobinuria and Aplastic
Anemia
By
Bénédicte Hugel,
Gérard Socié,
Thi Vu,
Florence Toti,
Eliane Gluckman,
Jean-Marie Freyssinet, and
Marie-Lorraine Scrobohaci
From the Institut d'Hématologie et d'Immunologie,
Faculté de Médecine, Université Louis Pasteur,
Strasbourg, France; the Laboratoire Central d'Hématologie,
Hôpital Saint-Louis, Paris, France; and the Unité de
Recherche sur la Biologie des Cellules Souches et Service de Greffe de
Moëlle, Hôpital Saint-Louis, Paris, France.
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ABSTRACT |
Paroxysmal nocturnal hemoglobinuria (PNH), frequently occurring
during suppressed hematopoiesis including aplastic anemia (AA), is a
clonal disorder associated with an increased incidence of thrombotic
events. Complement-mediated hemolysis, impairment of the fibrinolytic
system, or platelet activation are thought to be responsible for the
associated thrombotic risk. We investigated here the elevation of
membrane-derived procoagulant microparticles in the blood flow of such
patients. Elevated levels of circulating microparticles were in fact
detected in both de novo PNH patients and AA subjects with a PNH clone,
but not in those with AA without a PNH clone. The cellular origin of
the microparticles was determined in PNH samples; most stemmed from
platelets. Glycophorin A+ particles were rarely detected.
Therefore, platelet activation, resulting in the dissemination of
procoagulant phospholipids in the blood flow, could be one of the main
causes for the elevated thrombotic risk associated with PNH. These
observations suggest that shed membrane particles can be considered a
valuable biological parameter for the assessment of possible thrombotic
complications in patients with PNH.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PAROXYSMAL nocturnal hemoglobinuria (PNH)
is an acquired clonal disorder characterized by the presence of
abnormal hematopoietic cells deficient in
glycosylphosphatidylinositol (GPI)-anchored proteins.1
The association with suppressed hematopoiesis, including aplastic
anemia (AA), occurs frequently.2 The main clinical
manifestation is hemolytic anemia and the most common complications are
thrombosis, pancytopenia, and myelodysplastic syndrome or acute
leukemia.3 Although intravascular hemolysis can easily be
explained by a deficiency of GPI-anchored complement regulatory
proteins such as CD59 and CD55 on the membrane of red blood cells
(RBC),4 the mechanism responsible for the increased incidence of thrombotic events in PNH remains unclear. Apart from hemolysis itself, several failures of the fibrinolytic system have been
highlighted, including a deficiency of urokinase-type plasminogen
activator receptor on leukocytes presenting the PNH phenotype5 and increased plasma levels of soluble
urokinase-type plasminogen activator receptor.6
Furthermore, one patient with thrombotic complications in the course of
AA and/or PNH syndrome was shown to have
dysplasminogenemia.7 Other studies did not identify any
fibrinolytic defects but rather pointed to varying degrees of platelet
activation in PNH individuals.8 Beyond the increase of
expression of activation-dependent proteins, platelet stimulation is
accompanied by the loss of membrane phospholipid asymmetry. This
results in phosphatidylserine externalization and microvesicle
generation, as shown in an in vitro study.9 Furthermore,
transverse redistribution of plasma membrane phosphatidylserine and
cell fragmentation into phosphatidylserine-bearing microparticles is a
hallmark of cells undergoing apoptosis,10,11 and
phosphatidylserine becomes a determinant for phagocyte recognition of
senescent or apoptotic cells to be cleared.12,13 AA is
precisely a clinical situation in which programmed cell death is
thought to occur to a high degree.14
Membrane-derived microparticles have been shown to provide the
catalytic surface necessary for the assembly of the procoagulant enzyme
complexes, prothrombinase15 and tenase.16 In
the blood flow, the presence of high levels of procoagulant
microparticles, stemming from lysed RBC, apoptotic cells, or activated
platelets, could therefore be responsible for the dissemination of
prothrombotic seats. This prompted us to assess the increase of
circulating microparticles in the peripheral blood of AA and PNH
patients using insolubilized annexin V (AV), a protein showing a strong affinity for phosphatidylserine, through which capture was
feasible.11 Such circulating particles carry membrane
antigens specific for the cells they stem from and through which
capture was also achieved to determine their cellular origin.
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PATIENTS AND METHODS |
Patients.
Details of the patient clinical courses, treatment, and evolution have
been previously reported in detail.2,17 In brief, patients
were considered to have PNH if the Ham-Dacie's test was positive at
diagnosis. Patients with a previous history of AA who later developed a
positive Ham-Dacie's test and/or had evidence of defective expression
of GPI-linked proteins by flow cytometry were considered to have an
AA/PNH syndrome. Of the 29 patients with either PNH (n = 12) or an
AA/PNH syndrome, 2 patients (P1 and P4) had a history of thrombotic
complications. Patient P1 had PNH and developed a Budd-Chiari
syndrome.17 Patient P4, with an AA/PNH syndrome, had a
positive Ham-Dacie's test 4 years after diagnosis and evidence of a
deficiency in GPI-anchored proteins. Almost 10 years after being
treated by immunosuppressive therapy, she had thrombosis of the lower limb.
Materials.
The monoclonal antibody (MoAb) against glycophorin A was from
Immunotech S.A. (Marseilles, France). The MoAbs to human platelet glycoprotein Ib (GPIb) and glycoprotein IIIa (GPIIIa) were kind gifts from Dr F. Lanza (Unité 311 INSERM, Strasbourg, France). The irrelevant biotinylated Ig (IgG1Bi) was from Leinco
Technologies (Ballwin, MO). Purified human blood coagulation factors
were the same as those used in a recent study reported by our
group.18 Factor V was a product from Diagnostica Stago
(Asnières, France). Recombinant human annexin V was purchased from Euromedex (Souffelweyersheim, France) and conjugated with fluorescein isothiocyanate (FITC; annexin VFITC) following
the procedure described by Dachary-Prigent et al.19 High
binding capacity streptavidin-coated microtitration plates, 1-O-n-octyl- -D-glucopyranoside, biotin-X-OSu, and Chromozym TH were
from Boehringer Mannheim (Mannheim, Germany). Human serum albumin
(HSA), the streptavidin-R-phycoerythrin conjugate, phosphatidylcholine, and phosphatidylserine from bovine brain were products from Sigma Chemical Co (St Louis, MO). Calcium ionophore A23187,
D-phenylalanyl-prolyl-arginyl chloromethyl ketone (FPR.CK), and
1,5-dansyl-glutamyl-glycyl-arginyl chloromethyl ketone (Dns-EGR.CK)
were obtained from Calbiochem (San Diego, CA). All other reagents were
of the highest available purity grade.
Methods.
The preparation of platelet-free plasma samples, the biotinylation of
annexin V and MoAbs, the capture of microparticles by immobilized
annexin V or MoAbs, and the prothrombinase assay for the estimation of
the amount of captured microparticles are detailed in Aupeix et
al.11 It has to be mentioned that different incubation times were used for microparticle capture by annexin V (30 minutes) and
MoAbs (2 hours). However, to exclude that complement attack of cells
might occur during the blood drawing procedure and the separation of
plasma from cells by centrifugation, some PNH and control blood samples
were drawn into both EDTA and citrated anticoagulants. The two
successive centrifugation steps, requiring, respectively, 10 minutes
and 1 minute, were performed immediately, and plasma separated from
cells was processed for the determination of its microparticle
content.11 No difference was noticed between EDTA, commonly
used for complement assays, and citrated anticoagulant, normally used
for routine hemostasis assays, allowing us to perform the study with
citrated samples that were also used for the hemostasis follow-up of
the patients.
Flow cytometry.
RBC were analyzed by flow cytometry using a FACScan flow cytometer
(Becton Dickinson, San Jose, CA). The sheath fluid was Isoton II
balanced electrolyte solution (Coulter, Krefeld, Germany). Data
acquisition and analysis were conducted with the CellQuest software
(Becton Dickinson, San Jose, CA). Analysis of the ability of RBC to
undergo membrane vesiculation after stimulation by ionophore was
performed on 10,000 events per sample. Annexin VFITC was
used as a probe of phosphatidylserine exposure simultaneously for RBC
and derived microparticles.18 Glycophorin A labeling was
performed using the biotinylated antibody and the
streptavidin-R-phycoerythrin conjugate.
Functional detection of procoagulant phospholipid exposure in
stimulated cells and derived microparticles.
Procoagulant phospholipid exposure in stimulated RBC and derived
microparticles was investigated using a human prothrombinase assay in
which phosphatidylserine promotes the activation of prothrombin by
factor Xa in the presence of factor Va.20 Thrombin
generated by functional prothrombinase complex was measured using a
chromogenic assay as already described elsewhere.18 The
ability of RBC to expose phosphatidylserine and to release procoagulant
microparticles was examined after stimulation by 5 µmol/L calcium
ionophore A23187 for 90 minutes at 37°C in the presence of 2 mmol/L
external CaCl2. RBC were separated from derived
microparticles by centrifugation at 12,000g for 30 seconds
before measurement. In each case, results of PNH samples were compared
with the prothrombinase activities developed in counterparts from
healthy volunteers.
Statistical analysis.
Data are represented as the mean ± standard deviation (SD).
Statistical analysis was performed using the Student's two-tailed t-test or a variance comparison (according to the ratio method).
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RESULTS |
Capture and antigenic characterization of circulating particles in
blood samples from AA and PNH patients.
To assess one of the possible origins of thrombosis that frequently
occurs as a complication of PNH, we measured the levels of circulating
particles in peripheral blood of PNH individuals and control subjects
(Fig 1). AA blood samples were also assayed to explore the possible biological link between this disorder and PNH.
High to very high levels of circulating particles were indeed detected
in some PNH samples when compared with the control group (P < .00007). The mean ± SD for the control group was 5.3 ± 2.2 nmol/L phosphatidylserine equivalent, that of the aplastic anemia group
without a PNH clone (AA) was 1.9 ± 1.6 nmol/L, that of the aplastic
anemia with a PNH clone group (AA/PNH) was 11.1 ± 9.1 nmol/L, and
that of the PNH group 14.4 ± 10.5 nmol/L phosphatidylserine equivalent. Interestingly, a clear difference was established between
the two AA groups (P < .02). Some AA/PNH samples contained high microparticle levels, comparable with those of the PNH group, whereas AA samples were measured at lower values than the control group
(P < .0004).
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| Fig 1.
Amount of circulating microparticles in peripheral blood
samples from 26 control subjects, 8 AA individuals without a PNH clone
(AA), 17 AA individuals with a PNH clone (AA/PNH), and 12 PNH patients.
The particle capture procedure involving insolubilized AV as well as
the assay of their phosphatidylserine content based on the ability of
this phospholipid to promote the assembly of the clotting
prothrombinase enzyme complex are detailed in Aupeix et
al.11 The mean ± SD for the control group is 5.3 ± 2.2 nmol/L phosphatidylserine equivalent, that of the AA group is 1.9 ± 1.6 nmol/L, that of the AA/PNH group is 11.1 ± 9.1 nmol/L, and that
of the PNH group is 14.4 ± 10.5 nmol/L phosphatidylserine
equivalent. P values reflect the significance of the patients'
circulating particle levels compared with healthy controls or patients
from other indicated groups. Statistical analysis was performed using
the Student's two-tailed t-test.
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Therefore, we searched for a possible link between the level of the PNH
clone expression and the proportion of circulating particles. This was
performed taking into account the proportions of GPI-deficient
polynuclear cells, monocytes, platelets, or erythrocytes. Correlation
coefficients never exceeded 0.7. No clear correlation could therefore
be evidenced.
The cellular origin of these circulating particles was determined. No
significant amount of glycophorin A+ particles was captured
in most PNH samples (individuals having received recent blood
transfusions were, of course, excluded from the study), although PNH
RBC are believed to be one of the targets of the complement membrane
attack complex leading to hemolysis (Fig
2). However, the presence of glycophorin A at the surface of PNH RBC
was verified by flow cytometry analysis. The expression of glycophorin
A in PNH RBC was not impaired when compared with that of normal
erythrocytes, without or after stimulation by calcium ionophore (data
not shown). In contrast, very high levels of platelet-derived particles
bearing the GPIb-specific marker were captured in several PNH
samples. The antibody directed to GPIIIa yielded basically identical
results (data not shown). The means of the amount of GPIb+ particles were not significantly different between
PNH and control group at the .05 level, but the variances comparison
(using the ratio method) showed a clear difference in the distribution
of the values at the .002 level. This is consistent with a situation in
which some patients are in the range of the control group, whereas
others present very high levels of circulating GPIb+
particles. It has to be emphasized that no direct comparison between
capture by annexin V and antibodies could be performed because
preincubation times and affinities for the respective ligands are
different.11
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| Fig 2.
The cellular origin of circulating microparticles in
peripheral blood samples from PNH individuals and control subjects. The
capture procedure involved insolubilized MoAbs to human glycophorin A,
human GPIb , and irrelevant IgG1, the latter yielding control values
that never exceeded 3 nmol/L phosphatidylserine equivalent and were
subtracted from those reported in this figure. The assay of the
phosphatidylserine content of captured particles is based on the
ability of this phospholipid to promote the assembly of the
prothrombinase enzyme complex. Each value is the mean of triplicate
determinations. The means corresponding to the PNH samples were not
significantly different from their control counterparts at the .05 level using the Student's two-tailed t-test. Variances were
also compared (using the ratio method) and the distribution of the
values was shown to be different between the PNH and the control
samples at the .002 level.
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Assessment of phosphatidylserine exposure in individual PNH samples.
The ability of PNH RBC to externalize phosphatidylserine and to
vesiculate was examined in a functional prothrombinase assay with 4 PNH
samples: P1, P2, P3 (already used for the control of glycophorin A
expression), and P4. PNH RBC showed impaired procoagulant phospholipid
externalization in 2 cases (P2 and P4) among the 4 samples tested
(Fig 3). Vesiculation, assessed in the
supernatant of stimulated RBC, was almost undetectable in 3 (P2, P3,
and P4) among the 4 PNH RBC samples. Finally, in 1 PNH RBC sample (P1), procoagulant phospholipid externalization and membrane vesiculation appeared normal. It is of interest to notice that the absence of
activability of P2 RBC is probably related to its high basal stimulation state (see legend of Fig 3). This was not the case for P4
and P3 samples.
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| Fig 3.
Procoagulant phospholipid externalization and membrane
vesiculation in calcium ionophore-stimulated RBC from 4 PNH patients
(P) and 2 healthy subjects (T) measured by prothrombinase assay. Cells
were stimulated by 5 µmol/L calcium ionophore A23187 in the presence
of 2 mmol/L external CaCl2 for 90 minutes at 37°C.
Stimulated cells were centrifuged for 30 seconds at 12,000g.
Microparticle release was measured in the supernatant, whereas
phosphatidylserine externalization was measured on the pelleted cells.
Data (n = 3) represent the increase of prothrombinase
activity after stimulation and are expressed as the ratio between the
activity before and after ionophore stimulation. The basal activity of
the RBC before stimulation was 2.2, 4.5, 0.6, 0.2, 1.0, and 0.3 nmol/L
phosphatidylserine equivalent for P1, P2, P3, P4, T1, and T2,
respectively. In the supernatant, corresponding basal activities were
measured at 1.2, 1.3, 0.4, 0.2, 0.4, and 0.4 nmol/L phosphatidylserine
equivalent.
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To confirm the prothrombinase results, procoagulant phospholipids were
probed with AVFITC on stimulated PNH and control RBC and on
derived microparticles from the same individuals
(Fig 4). The flow cytometry analysis showed
lower to moderate levels of procoagulant microparticles in the
supernatant of ionophore-stimulated PNH RBC compared with control RBC.
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| Fig 4.
Flow cytometry analysis of the shedding of membrane
microparticles from PNH (P) and control (T) RBC after calcium ionophore
treatment. Dot plot representations of AVFITC-labeled cell
and particle suspensions. Cells have the highest forward scatter signal
(FSC), whereas derived microparticles have a lower one. The proportion
of events in each gate is indicated. Stimulation was achieved by 5 µmol/L calcium ionophore A23187 in the presence of 2 mmol/L external
CaCl2 for 90 minutes at 37°C. Fluorescence intensity
reflects the extent of AVFITC labeling of the population of
interest, testifying to the degree of phosphatidylserine
externalization. Each dot plot corresponds to 10,000 events and is
representative of three experiments performed likewise.
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Finally, 3 PNH RBC samples of 4 (P2, P3, and P4) showed an impaired
vesiculation in a functional prothrombinase assay (Fig 3) and 3 PNH RBC
samples of 3 (P1, P2, and P3) were unable to shed normal levels of
particles as deduced from flow cytometry analysis (Fig 4). P1 sample
led to apparent contradictory results. The proportion of shed membrane
microparticles after stimulation was lower than in controls when
measured by flow cytometry, but the functional prothrombinase assay did
not show any vesiculation impairment.
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DISCUSSION |
The present study clearly demonstrates the presence of elevated levels
of phosphatidylserine-bearing microparticles in the peripheral
circulation of several PNH patients or subjects with AA with a PNH
clone. The procoagulant potential disseminated by these particles in
the blood flow could be responsible, at least in part, for the high
incidence of associated thrombotic complications. The basal level of
circulating particles detected in control subjects probably reflects a
balance between cell proliferation, stimulation, and death and concerns
microparticles that transiently escape destruction by
phagocytosis,13 phospholipases,21 or
confinement by specific adhesion.22 In pathological
situations in which apoptosis or cell stimulation is known to occur at
a high degree, the elimination systems could be saturated, giving rise
to increased levels of circulating shed microparticles. Interestingly,
samples from AA patients who did not develop a PNH clone were measured under the control level. Therefore, the susceptibility of RBC and
platelets lacking GPI-linked complement inhibitors of the membrane
attack complex may account for the high levels of shed microparticles.
But again, the proportion of circulating microparticles is probably
dependent on the relative efficiency of clearance systems. Hence,
individual variability of the elimination response may account for the
lack of clear correlation between the PNH clone level and the particle proportion.
Very low amounts of particles bearing the RBC marker glycophorin A were
captured in PNH plasmas, whereas hemolysis often occurs during the
course of PNH. The normal expression of glycophorin A on PNH RBC and
the specificity of the corresponding antibody were controlled because
of the qualitative abnormality of glycophorin A reported by Parker et
al.23 The circulating microparticles did actually not
originate from lysed RBC to a significant extent. On the other hand,
very high levels of particles of platelet origin were detected in
several PNH samples. Hence, platelet activation, already reported in
PNH,8,9,17 could be one of the main causes of the high
incidence of thrombosis associated with PNH. It has to be emphasized
that platelet-derived microparticles were easily detectable in control
samples and probably account for an important part of the basal
particle level.
The absence of RBC-derived particles in PNH samples led us to
investigate the ability of RBC to externalize phosphatidylserine and to
vesiculate. An impaired ability of RBC to vesiculate has already been
reported by Whitlow et al24 for 2 PNH patients lacking CD59
and CD55. We also observed a very weak ability of some PNH RBC to
vesiculate using a functional prothrombinase assay, and we assume that
it could explain the absence of circulating RBC-derived microparticles.
The heterogeneity of the responses of the RBC samples to ionophore is
probably linked to the random selection of the patients. The different
stages of evolution of the disease and current treatments might, at
least in part, explain such an heterogeneity. The impaired ability to
vesiculate might also be related with the resistance to apoptosis
observed in PNH granulocytes.25
The use of the prothrombinase assay concomitantly with flow cytometry
showed apparent contradictory results with the RBC sample P1.
Vesiculation was lower than in control samples when measured by flow
cytometry, but normal in a functional prothrombinase assay. This
observation points to the fact that the two analyses do not measure the
same parameters. Flow cytometry enables us to estimate the proportion
of particles, whereas the prothrombinase assay detects their
procoagulant potential. Therefore, the P1 RBC actually show an impaired
ability to vesiculate, but the microparticles shed in the supernatant
might bear an increased procoagulant potential. Interestingly, patient
P1 recently experienced several thrombotic events. On the other hand,
P4 RBC showed impaired ability to externalize phosphatidylserine and to
vesiculate by prothrombinase assay, but patient P4 also developed thrombosis.
Whitlow et al24 also evidenced an impaired ex vivo
vesiculation of platelets completely lacking GPI-linked proteins,
whereas Wiedmer et al9 were able to induce
complement-mediated platelet membrane vesiculation in platelet samples
lacking CD59 antigen. Here, we have detected very high levels of in
vivo circulating platelet-derived microparticles in several PNH blood
samples. In our case, platelet vesiculation was not directly
investigated using isolated platelets. It can be reasonably assumed
that our assay measures a balance between the shedding of particles in the blood flow and elimination by the various clearance systems. Therefore, it is conceivable that significant levels of particles of
platelet origin are consistently released in the blood flow, although
higher or equivalent amounts of particles of RBC origin are shed, but,
for unknown reasons, the latter might be more efficiently eliminated
from the circulation. The description by Simons and Ikonen26 of functional sphingolipid-cholesterol rafts in
cell membranes might explain such differences, especially in a context of absence of GPI-anchored proteins. CD55 and CD59 have been precisely shown to be sorted and shed in exosomes during reticulocyte
maturation.27
Circulating particles, giving rise to disseminated potential
prothrombotic seats, are certainly not neutral with respect to the
response of the coagulation system of an individual. In several PNH
patients investigated, these particles seem to originate massively from
platelets and their amount could be responsible in part for thrombotic
events. Moreover, platelet microparticles released after complement
activation were shown to be enriched in the membrane receptor for
coagulation factor Va,15 the latter involving
phosphatidylserine.28
On a therapeutic point of view, the measurement of the proportion of
circulating microparticles can be of interest with regard to the choice
of the anticoagulant treatment. The balance between hemorrhage and
thrombosis is believed to be subtle in the PNH pathology. It is
probably related to the frequent association of pancytopenia with
severe cell activation. Therefore, if high levels of circulating
microparticles increase the thrombotic tendency, anti-vitamin K
treatment should be the more appropriate preventive approach. Moreover,
PNH patients with pancytopenia and high levels of circulating
microparticles should not be at risk of bleeding episodes, whereas PNH
patients with pancytopenia but low levels of circulating microparticles
might probably be. Elevated levels of circulating platelet
microparticles were precisely found protective against bleeding in
patients with autoimmune thrombocytopenia but were associated with the
occurrence of small cerebral vessel infarcts when very
high.29 Helpful indications can be deduced from the new
biological parameter consisting of the level of circulating procoagulant microparticles. This system may enable us to assess an
instant in vivo thrombotic risk associated with PNH owing to the
possibility of massive release of procoagulant microparticles shed from
activated platelets in the blood flow. However, it would be interesting
to investigate both circulating microparticles and other coagulation
parameters in more homogeneous groups of patients who previously
developed thrombosis to evaluate the multifactorial character of the
mechanisms of thrombosis in the PNH pathology. But, the relative rare
occurrence of this disorder is certainly a limit for such studies.
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FOOTNOTES |
Submitted June 16, 1998; accepted January 13, 1999.
Supported by Grants from the Institut National de la Santé et de
la Recherche Médicale, the Université Louis Pasteur de Strasbourg, and the Fondation pour la Recherche Médicale.
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 Marie-Lorraine Scrobohaci, MD, Laboratoire
Central d'Hématologie, Hôpital Saint-Louis, 1, avenue
Claude Vellefaux, 75475 Paris Cedex 10, France.
| |
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