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THE THROMBOTIC THROMBOCYTOPENIC PURPURA AND HEMOLYTIC UREMIC SYNDROMES: NEW INSIGHTS AND NEW TREATMENTS

Role of ADAMTS13 in the pathogenesis, diagnosis, and


treatment of thrombotic thrombocytopenic purpura
Johanna A. Kremer Hovinga1 and Bernhard Lammle1
1Department

of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital and
the University of Bern, Bern, Switzerland

The regulation of VWF multimer size is essential in preventing spontaneous microvascular platelet clumping, a central
pathophysiologic finding in thrombotic thrombocytopenic purpura (TTP). In the majority of TTP patients, ADAMTS13,
the principal regulator of VWF size, is severely deficient. Today, 2 forms of severe ADAMTS13 deficiency are
recognized. The acquired form is caused by circulating autoantibodies inhibiting ADAMTS13 activity or increasing
ADAMTS13 clearance. Pathogenic anti-ADAMTS13 Abs are mainly of the IgG class, predominantly of subclass IgG4,
and inhibitory Abs recognize a defined epitope in the ADAMTS13 spacer domain. The reasons underlying the failure to
maintain immunologic tolerance to ADAMTS13, however, are still poorly understood. Constitutional ADAMTS13
deficiency leading to hereditary TTP, also known as Upshaw-Schulman syndrome, is the result of homozygous or
compound heterozygous ADAMTS13 gene mutations.

Introduction
Thrombotic thrombocytopenic purpura (TTP) became recognized in
1947, when Singer et al reviewed all 12 cases known at the time,
including Moschcowitzs patient, for similarities and differences
from other forms of thrombocytopenic purpura.1 The name was
ingenious because it not only described the histopathologic finding
of generalized arteriolar and capillary platelet thrombosis, but also
suggested the most likely pathophysiologic mechanism underlying
thrombocytopenia: the consumption of platelets by the formation of
innumerable platelet thrombi. The investigators concluded that an
appropriate study on the pathogenesis of TTP would only become
possible with appropriate disease recognition ante mortem. In the
late 1970s, the first reports of successful treatment of acute TTP
episodes with plasma exchange or plasma infusions emerged.
Among these early reports was the excellent description by Jefferson D. Upshaw of the clinical course of a female patient who
presented with recurrent microangiopathic hemolysis and thrombocytopenia, which were reverted when the patient received plasmacontaining blood products.2 Upshaw concluded that his patient was
similar to a patient described by Schulman et al almost 20 years
previously,3 and that both suffered from a congenital deficiency of a
plasma factor.
The search for the pathogenesis underlying TTP gained momentum
when Moake et al observed the presence of unusually large VWF
multimers in the plasma of 4 patients,4 including those of Schulman
et al3 (as patient B) and Upshaw2 (as patient D), with a chronic
relapsing course of TTP in remission. Moake et al hypothesized that
the unusually large VWF, similar in size to VWF multimers in the
supernatant media of endothelial cell cultures, were the consequence of a lacking depolymerase and accountable for in vivo
platelet clumping in the microvasculature observed in TTP.4 The
identification of the peptide bond cleaved during physiologic
processing of VWF (Tyr1605-Met1606 in the VWF A2 domain)
paved the way for the identification of the VWF-cleaving protease
by Furlan et al5 and Tsai.6 In 1997, the first link between a chronic
relapsing course of TTP, the presence of unusually large VWF
multimers in plasma in remission, and missing VWF-cleaving

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Figure 1. Pathogenesis of TTP. Shown are endothelial cell cultures


stimulated to release VWF multimers perfused with fluorescence-labeled
platelets suspended in a plasma with a severe ADAMTS13 deficiency
of 5% of the normal (top panel) or in a plasma with normal
ADAMTS13 activity (bottom panel). Experiments were performed by
Dr R. Frohlich-Zahnd in the laboratory of Prof J.-F. Dong (at the time with
the Department of Medicine, Baylor College of Medicine, Houston, TX,
and now at the Puget Sound Blood Center, Seattle, WA).47,48

protease activity was established.7 Shortly thereafter, a severe


VWF-cleaving protease deficiency caused by circulating IgG autoantibodies inhibiting the protease was found in the majority of adult
patients suffering from acquired TTP.8,9 During the ensuing years,
the VWF-cleaving protease was purified from plasma and subjected
to N-terminal amino acid sequence analysis,10,11 mapped to chromosome 9q34, cloned, and identified as the 13th member of the
ADAMTS (a disintegrin and metalloprotease with thrombospondin
repeats) family of metalloproteases.12-14 Patients with hereditary
TTP were found to carry ADAMTS13 mutations.12
Today, 2 forms of severe ADAMTS13 deficiency ( 5% of the
normal amount) are recognized: an acquired form and a congenital
form, both leading to sustained VWF-dependent accumulation of
platelets in small vessels that eventually results in microvascular
thrombosis and TTP (Figure 1).

Acquired ADAMTS13 deficiency


Circulating anti-ADAMTS13 Abs are found in plasma of nearly all
(94%-97%) patients suffering from idiopathic TTP and severe

American Society of Hematology

Figure 2. Schematic protein domain structure of ADAMTS13 and localization of anti-ADAMTS13 autoantibody epitopes. The ADAMTS13
protein consists of 1427 amino acid residues and the domain structure includes a signal peptide (S) and a pro-peptide (P), a metalloprotease, a
disintegrin (Dis), a cysteine-rich (Cys), a spacer, and 2 CUB domains, as well as 8 thrombospondin type 1 repeats (1-8) (top row). Bottom rows (A-C):
Lines below ADAMTS13 depict the ADAMTS13 fragments used for epitope mapping of anti-ADAMTS13 Abs. Y indicates that Abs directed against the
respective ADAMTS13 fragment were found in a certain proportion of patients investigated (percentage indicated). In all 3 studies (A-C), only patients
suffering from acute TTP with severe acquired ADAMTS13 deficiency were investigated. Row A: Klaus et al investigated 25 patients using
E coli derived recombinant ADAMTS13 fragments.17 Row B: Luken et al performed epitope mapping in 7 patients using full-length ADAMTS13 and
ADAMTS13 fragments produced in insect cells.18 Row C: Zheng et al performed epitope mapping on 67 patient samples using rADAMTS13 and
rADAMTS13 fragments produced in mammalian cell lines.19

acquired ADAMTS13 deficiency. In the majority of cases, these


Abs inhibit ADAMTS13 activity, but 10%-15% of patients have
noninhibitory Abs, and severe ADAMTS13 activity is thought to be
the result of an increased Ab-mediated clearance. The Abs are
mainly of the IgG isotype, predominantly of subclass IgG4, followed by IgG1, IgG2, and IgG3.15 High levels of IgG4 were found in
relapsed cases of TTP and were associated with an increased risk of
relapse,15 whereas the presence of IgA and/or IgG1 at presentation
was associated with adverse outcome in a small number of
patients.15,16
ADAMTS13 Abs recognizing a primary epitope on the ADAMTS13
spacer domain are present in 97%-100% of patients with severe
Ab-mediated ADAMTS13 deficiency, and up to 64% of these
patients also have Abs that recognize epitopes in other ADAMTS13
domains (Figure 2).17-19 Fine-mapping of the ADAMTS13 spacer
domain epitope identified the amino acids Arg568, Phe592, Arg660,
Tyr661, and Tyr665 as the primary antigenic target of inhibitory
ADAMTS13 Abs.20-22 As yet, the epitopes of ADAMTS13 Abs
binding to other ADAMTS13 domains have not been identified.
The mechanisms leading to loss of self-tolerance and induction of
autoimmunity in acquired TTP are still poorly understood (for a
detailed review, see Pos et al23). As in other autoimmune diseases,
microbial agents have been implicated in the onset of acquired TTP.
Given the large variety of microbial agents suspected to play a
pathophysiologic role in TTP, it is difficult to assess whether these
are indeed causative triggers or whether the associations described
represent a publication bias due to the lasting interest in this rare
disease (annual incidence, 1.72 cases/million24) with a steadily
increasing number of annual publications (Figure 3). In this review,

Hematology 2012

we emphasize 3 cases of proven enterohemorrhagic Escherichia coli infection and typical hemolytic uremic syndrome
(DHUS), in which ADAMTS13 activity is usually normal or only
mildly reduced. In a study of 29 children with DHUS, one was
found to have a severe deficiency of ADAMTS13 activity, which
normalized in remission.25 During the German E coli O104:H4
outbreak in 2011, plasma samples of 6 patients with severe clinical
presentation, including neurologic symptoms, were referred to our
laboratory for ADAMTS13 activity determination. Two of these
6 patients had a severe acquired ADAMTS13 deficiency due to
inhibitory autoantibodies of IgG isotype. One of the 2 patients has
since experienced a relapse of thrombotic microangiopathy, displaying again a severe acquired ADAMTS13 deficiency and inhibitor
(Prof B. Potzsch, Experimental Hematology and Transfusion Medicine, University of Bonn, Germany; personal communication).
Whether these 3 DHUS cases were unfortunate to suffer from
2 rare diseases (acquired TTP with severe ADAMTS13 deficiency
and DHUS) at the same time, or if enterohemorrhagic E coli in
certain circumstances can trigger the formation of anti-ADAMTS13
autoantibodies, or if Shiga toxin, in analogy to the situation in
ADAMTS13 knockout mice,26 was a trigger in patients with so far
asymptomatic autoantibody-mediated ADAMTS13 deficiency remains to be elucidated.
Evidence for a genetic predisposition to developing pathogenic
ADAMTS13 Abs comes from the following 3 observations. First,
the MHC class II allele HLA DRB1*11 was found to be overrepresented in patients suffering from acute TTP with severe acquired
ADAMTS13 deficiency in France, the United Kingdom, and
Germany.27-29 Conversely, the HLA DRB1*04 allele frequency was

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Figure 3. Investigator fascination with TTP. Shown are publications retrieved from PubMed using the search term thrombotic thrombocytopenic
purpura. Given the annual incidence of TTP (idiopathic TTP, 4.5 cases/million; idiopathic TTP with severe ADAMTS13 deficiency, 1.72 cases/
million24), there seem to exist almost more publications than cases. PEX indicates plasma exchange. The pentad of clinical signs of TTP was described
by Amorosi and Ultmann,51 the Canadian plasma exchange study was reported by Rock et al.52

higher in healthy controls in all 3 populations, hinting at a protective


effect of this MHC class II allele. Second, the report on identical
twin sisters not carrying the HLA DRB1*11 allele who developed
acute acquired TTP with severe ADAMTS13 deficiency due to
inhibitory autoantibodies 14 months apart30 suggests additional,
thus far unrecognized genetic risk factors. Third, a considerable
number (11% and 9.6%, respectively) of heterozygous carriers of
ADAMTS13 mutations were found among patients diagnosed with
acute acquired TTP and severe ADAMTS13 deficiency.31,32 Seven
mutation carriers of these 2 reports had ADAMTS13 Abs of the IgG
isotype, which were shown in 6 patients to be functional ADAMTS13 inhibitors. Given the low prevalence of ADAMTS13
mutations in the population, the number of heterozygous mutation
carriers among TTP patients with severe acquired ADAMTS13
deficiency is remarkable and suggests a role of heterozygous
sequence variants either in the induction of autoantibodies or in
facilitating an ADAMTS13 decrease below a threshold relevant for
the induction of TTP.
A role of T cells in the deregulated immune response in acquired
TTP is likely, but has not yet been formally investigated. In addition
to the MHC class II link, somatic hypermutation observed in
ADAMTS13 mAbs derived from peripheral B cells and from
switched memory B cells isolated from the spleen suggests a role
for helper T cells during ADAMTS13 autoantibody formation.18,33
Further evidence comes from the clinical observation during plasma
exchange therapy that 30%-50% of TTP patients with severe
acquired ADAMTS13 deficiency experience a clinical exacerbation
that is often associated with an increase of ADAMTS13 Ab and
functional inhibitor titers, a phenomenon apparently not observed in
patients treated with cyclosporine A, a T-cell immunosuppressant.34

Congenital ADAMTS13 deficiency and hereditary TTP


Hereditary TTP (Upshaw-Schulman syndrome [USS]2,3) due to
severe congenital ADAMTS13 deficiency is the result of compound
heterozygous or homozygous ADAMTS13 gene mutations. More
than 140 different ADAMTS13 mutations, which include missense

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mutations (approximately 60%), small deletions and insertions


(approximately 20%), as well as nonsense and splice site mutations,
have been identified in affected patients thus far. There is a
considerable genetic heterogeneity, with the majority of mutations
confined to single families; patients with homozygous mutations are
found mainly in families with consanguinity. Two mutations stand
out: the single base insertion 4143insA in exon 29 and the missense
mutation Arg1060Trp in exon 24 have both been observed in
several unrelated families over a wide geographic area. While the
single base insertion 4143insA seems to cluster around the Baltic
sea in Scandinavia and Moravia,35 the Arg1060Trp mutation has
been observed in the United States and in many countries across
Europe.32,36
Worldwide, more than 150 patients diagnosed with USS are alive.
Available published data seem to confirm earlier findings of an
age-dependent clustering of the first TTP attack, with half of these
patients presenting with a first acute TTP bout within their first years
of life (early onset). The other half seem to remain asymptomatic
into early adulthood and to suffer from a first acute attack between
20 and 40 years of age, and a few patients are even asymptomatic
until they are 60 years of age (late onset).37,38 Regardless of the
age at disease onset, once a first TTP bout has occurred, USS
patients often present with a chronic relapsing course, recurrences
of acute episodes often being triggered by events such as pregnancy
or heavy alcohol intake.36,39 In addition to age at onset, the clinical
presentation in USS patients is variable and may even differ in
families and in unrelated patients bearing the same 2 ADAMTS13
mutations. However, patients with neurologic involvement also tend
to present with neurologic symptoms during subsequent episodes,
whereas others may never present with neurologic findings but
suffer from severe renal impairment instead. For this reason,
establishing a link between the ADAMTS13 genotype and the
clinical phenotype in USS is difficult. It was reported recently that
residual ADAMTS13 activity in the range of 0.5%-6.8%, as
determined by a 73amino acid VWF peptide assay, influenced the
age of disease onset and episode frequency.38 The prevalent

American Society of Hematology

ADAMTS13 mutation Arg1060Trp is particularly frequent in USS


patients presenting with a first acute TTP episode during a first
pregnancy.32,36 Moreover, the clinical phenotype may also be
influenced by defects in other genes, as reported in an Italian USS
family in which one of the affected siblings with predominant renal
involvement had a complement factor H mutation in addition to her
2 ADAMTS13 mutations.40
Acute episodes can be prevented by regular plasma infusions every
2-3 weeks, although not all patients seem to require prophylaxis
because they experience relapses only in situations of increased risk
(eg, pregnancy and alcohol binge drinking).38,39 The challenge is to
define which patients should be advised to receive regular prophylactic plasma infusions to prevent long-term neurologic and
renal sequelae. This is particularly difficult in patients presenting
with an adult onset and only infrequent acute episodes, although
the latest experimental evidence from an animal model suggests
that all hereditary TTP patients may benefit from regular
ADAMTS13 replacement because the absence of ADAMTS13
seems to be associated with accelerated atherosclerosis.41 Longterm follow-up data on hereditary TTP cases are therefore urgently
needed and constitute a primary goal of the hereditary TTP registry
(www.ttpregistry.net; www.clinicaltrials.gov registration number
NCT01257269).
The observed plasma half-life of circulating ADAMTS13 (2-3 days)
and the empirically determined suitable interval between prophylactic plasma infusions of 2-3 weeks in most patients is discrepant and
suggests as yet unknown modes of action or storage reservoirs of
infused ADAMTS13. Apart from incidental allergic reactions to
infused plasma, supplementation with exogenous ADAMTS13 is
generally well tolerated. So far, no alloimmunization with the
occurrence of inhibitory ADAMTS13 Abs has been documented in
frequently transfused USS patients, although fluctuating titers of
anti-ADAMTS13 Abs of IgG isotype measured by ELISA have
been observed in several cases.39,42 The significance of these Abs is
unknown, especially because recovery and plasma half-life of
infused ADAMTS13 were normal in one of these patients.42 Despite
the strong pathogenic immune response seen in acquired TTP,
plasma-derived ADAMTS13 per se thus seems to have little or no
immunogenicity in USS patients. This is in clear contrast to
hemophilia A, in which one-quarter to one-third of factor VIII
treated patients develop inhibitory alloantibodies; induction of
immune tolerance is an important issue in hemophilia care.
USS is considered an extremely rare disorder. Reliable prevalence
and incidence data are missing and frequencies have been based on
the number of diagnosed cases in certain areas or on the proportion
of hereditary ADAMTS13 deficiency of the whole cohort of severe
ADAMTS13-deficient patients in a certain period of time. The large
geographic distribution areas of the ADAMTS13 mutations, 4143insA
and Arg1060Trp,32,35 the occurrence of homozygous mutations in
unrelated families,32,35,38,39 the notion that hereditary TTP is not only
a disease of infancy or childhood, and the increasing number of
patients recognized with an adult onset of disease suggest that the
prevalence of USS, although it remains an orphan disease, may have
been underestimated. This is corroborated by the first population
screening carried out in Japan, where heterozygous mutations were
present in a considerable number of healthy adults stratified
according to their plasma ADAMTS13 activity.43 The investigators
estimated that the true prevalence of USS could be up to 3-fold
higher than the number of patients diagnosed thus far in Japan.

Hematology 2012

ADAMTS13 and diagnosis and treatment of TTP


The first retrospective studies found a severe ADAMTS13 deficiency in 87%-100% of patients suffering from idiopathic TTP.8,9
Subsequent investigations of less selected cohorts of patients
diagnosed clinically with idiopathic TTP reported lower frequencies
of severely ADAMTS13-deficient patients (33%-80%). These studies, however, showed that severe ADAMTS13 deficiency was
extremely rare in secondary forms of thrombotic microangiopathies,
such as those after stem cell transplantation or associated with
neoplasia or drugs.44
Although a severe deficiency of ADAMTS13 is not found in all
patients diagnosed with idiopathic TTP, ADAMTS13 assays may
provide helpful guidelines for treatment. A finding of a severe
autoantibody-mediated ADAMTS13 deficiency provides the rationale for adjunctive steroids or treatment escalation with rituximab
or splenectomy in plasma refractory or relapsed cases, as well as
prognostic information regarding survival and risk of relapse.
Nearly half of the survivors initially presenting with an ADAMTS13
activity level 10% will eventually relapse, with the highest risk in
the first 2 years after an acute episode, whereas patients without this
deficiency rarely relapse.44 All patients with a prior history of
idiopathic TTP due to severe acquired ADAMTS13 deficiency will
have severe acquired ADAMTS13 deficiency in a subsequent
relapse. Therefore, a severe ADAMTS13 deficiency at any time
during remission is associated with an increased risk of relapse,45
although the finding of an ADAMTS13 value 10% of the normal
level is not necessarily a sign of an imminent relapse.44
Citrated plasma (also suitable are serum or heparinized plasma)
should be withdrawn for ADAMTS13 testing before the initiation of
treatment. Since the identification of the VWF-cleaving protease,
several different ADAMTS13 activity assays have been developed.
Initially, plasma-derived or recombinant multimeric VWF served as
a substrate and required the use of low ionic strength and high
concentrations of denaturing substances to unfold globular VWF to
make the peptide bond Tyr1605-Met1606 in the VWF A2 domain
accessible to cleavage by ADAMTS13. These assays were laborious, had turnaround times of 2-4 days, and were restricted to a few
specialized laboratories. Newer, second-generation assays use a
VWF A2 domain peptide of 73 amino acids identified as the VWF
minimal substrate for ADAMTS1346 or a somewhat larger derivative thereof. These assays, including several commercial assays, are
easy to perform, robust, and have short turnaround times. The latter
is important because getting patient samples to laboratories offering
these assays has become the time-limiting factor. Agreement
between assays using multimeric VWF substrate and assays using
VWF A2 domain peptides as substrate is generally good, but there
are some patients in whom a severe ADAMTS13 deficiency by one
assay may be associated with an only moderately or mildly reduced
ADAMTS13 activity by the other assay.44 It should be kept in mind
that even present-day peptide substrate assays do not reflect the
short interaction time of ADAMTS13 and multimeric VWF in vivo.
Therefore, a normal or mildly reduced ADAMTS13 activity by
contemporary assays does not necessarily preclude a causal role of
ADAMTS13 in a patients clinical scenario. Exemplary to this
phenomenon is the remarkable course of a patient over 8 years and
6 acute TTP episodes.47 Despite anti-ADAMTS13 Abs demonstrable by ELISA, dot blot, and immunoprecipitation during all
6 episodes, ADAMTS13 activity was normal by 3 different assays
in his first episode. During subsequent acute TTP episodes, severe
ADAMTS13 deficiency was observed first by a flow-chamber
assay, which gets close to the in vivo situation,48 and in later

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episodes by an initial rate assay using a peptide substrate, and finally


by a static end-point assay using multimeric VWF substrate.47
Caution is thus warranted when using in vitro ADAMTS13
activity 10% as a single criterion for the distinction between
idiopathic TTP and other thrombotic microangiopathies.
ADAMTS13 inhibitors are assessed by classical mixing studies of
heat-inactivated patient plasma and a normal plasma pool. Results
are expressed as Bethesda units per milliliter. In addition, antiADAMTS13 Abs can be detected by Western blotting or ELISA
using recombinant ADAMTS13 as antigen. In contrast to research
assays, many of the commercial ELISAs that are now widely used
detect ADAMTS13 Abs in 13%-15% of healthy controls, limiting
the utility of these assays to distinguish hereditary from acquired
ADAMTS13 deficiency or to identify patients at risk of imminent
recurrence after an acute episode of acquired TTP.

Conclusions
The fascination with TTP, a rare but exemplary disease, has
remained high over several decades (Figure 3) and recombinant
ADAMTS13, which will be available in the near future, will
add to this fascination. This will change the life of patients
diagnosed with USS and possibly also have an impact on the
treatment of patients diagnosed with acquired idiopathic TTP, in
whom neutralization of functional inhibitors with recombinant
ADAMTS1349 or, even better, with a recombinant ADAMTS13
gain-of-function variant,50 is feasible, at least in vitro. Recombinant ADAMTS13 may also become useful for other diseases in
which extensive interaction between VWF and platelets plays an
imminent pathophysiologic role, such as myocardial infarction or
cerebrovascular stroke, which are not seldom important features
in acute TTP episodes.

Acknowledgments
The authors thank Monica Schaller, Irmela Sulzer, Gabriela Mader,
Carlo R. Largiade`r, Sara C. Meyer, Rahel Frohlich-Zahnd, Jan-Dirk
Studt, and Magnus Mansouri Taleghani for work in our laboratory
and the members of the Steering Committee of the Hereditary TTP
Registry and our long-term collaborators for their continued support
and input (in alphabetical order): Ulrich Budde, Hamburg, Germany; Jing-Fei Dong, Seattle, WA; Tobias A. Fuchs, Boston, MA;
Yoshihiro Fujimura, Nara, Japan; Miha Furlan, Bern, Switzerland;
James N. George, OK; Ingrid Hrachovinova, Prague, Czech Republic; Anne-Sophie von Krogh, Trondheim, Norway; Masanori
Matsumoto, Nara, Japan; Toshiyuki Miyata, Osaka, Japan; Petter
Quist-Paulsen, Trondheim, Norway; Barbara Plaimauer, Vienna,
Austria; Inge Scharrer, Mainz, Germany; Fritz Scheiflinger, Vienna,
Austria; Reinhard Schneppenheim, Hamburg, Germany; Deirdra R.
Terrell and Sara K. Vesely, OK; Jan Voorberg, Amsterdam, The
Netherlands; Denisa D. Wagner, Boston, MA; and X. Long Zheng,
Philadelphia, PA. Due to space constraints we were unfortunately
not able to cite every relevant paper.
Our research is supported by grants from the Swiss National Science
Foundation (32003B-124892), the International Society on Thrombosis & Haemostasis 2007 Presidential Fund, and by Baxter
Healthcare for our hereditary TTP registry.

Disclosures
Conflict-of-interest disclosure: J.A.K.H. and B.L. have each received research funding from and consulted for Baxter Healthcare.
Off-label drug use: None disclosed.

614

Correspondence
J. A. Kremer Hovinga, MD, Department of Hematology and
Central Hematology Laboratory, Inselspital, Bern University
Hospital and the University of Bern, CH-3010 Bern, Switzerland; Phone: 41-31-632-90-22; Fax: 41-31-632-18-82; e-mail:
johanna.kremer@insel.ch.

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