Received: 27 February 2020 Revised: 7 May 2020 Accepted: 11 May 2020

DOI: 10.1111/hae.14561

SUPPLEMENT ARTICLE

Diagnosis of rare bleeding disorders

Karina Meijer1 Waander van Heerde2,3 Keith Gomez4

1
Division of Thrombosis and Haemostasis,
Department of Haematology, University Abstract
Medical Centre Groningen, University of
Rare bleeding disorders result in significant morbidity but are globally underdiag-
Groningen, Groningen, The Netherlands
2 nosed. Advances in genomic testing and specialist laboratory assays have greatly
Radboud University Medical Center,
Hemophilia Treatment Centre, increased the diagnostic armamentarium. This has resulted in the discovery of new
Nijmegen-Eindhoven-Maastricht, Nijmegen,
genetic causes for rare diseases and a better understanding of the underlying molec-
The Netherlands
3
Enzyre, Nijmegen, The Netherlands
ular pathology.
4
Haemophilia Centre and Thrombosis Unit,
KEYWORDS
Royal Free London NHS Foundation Trust,
London, UK blood coagulation disorders, blood platelet disorders, hemorrhagic disorders, whole exome
sequencing, whole genome sequencing
Correspondence
Keith Gomez, Haemophilia Centre and
Thrombosis Unit, Royal Free London NHS
Foundation Trust, Pond Street, London NW3
2QG, UK.
Email: k.gomez@ucl.ac.uk

This article was originally published in

Haemophilia 2021 State of the Art: WFH
Virtual Summit, 14th-19th June 2020 Issue
Volume 27 Issue S3
(https://doi.org/10.1111/hae.14049). This
version includes a visual abstract from the
authors.

1 INTRODUCTION particularly those with a milder phenotype, which is discussed in this

In the 2018 global survey of the World Federation of Haemophilia,
14% of patients are recorded as having bleeding disorders other than
haemophilia or von Willebrand disease (wfh.org). This figure is a signif-
icant increase on the 9% recorded in the 1999 survey. However, in the
2019 report from the UK bleeding disorders registry the percentage
of patients with other bleeding disorders is 44% (ukhcdo.org). This
indicates that the use of the term ‘rare’ to collectively describe these
disorders may be appropriate on a global scale but may be somewhat of
a misnomer in developed healthcare systems. In fact, the commonest
of these ‘rare’ disorders, factor XI deficiency, is slightly more prevalent
in the UK (13% of all registered patients) than haemophilia A (12%)
and much more common than haemophilia B (3%). This reflects the rel-
atively high proportion of Jews in the UK and also includes registration
of mild heterozygous cases. These figures also suggest that on a global
scale, there is a significant underdiagnosis of rare bleeding disorders,
review.
It is a well-known limitation of the work-up for a suspected bleed-
ing disorder that we cannot provide a diagnosis to every patient. Partly,
this is caused by an imperfect referral process. Sometimes, it is dif-
ficult for non-experts to differentiate between abnormal and normal
bleeding patterns and a significant proportion of referred patients have
no clearly increased bleeding tendency, when evaluated by an expert
physician. However, there is also a large group of patients with a clear
suspicion of a bleeding disorder who do not receive a definite diagnosis.
To define increased bleeding tendency, contemporary studies most
often use a Bleeding Assessment Tool (BAT). The recent European
Haematology Association (EHA) Consensus Report uses a cut-off of ≥4
for males and ≥6 for females.1 In recent cohorts, many patients are
included with lower levels. In a Dutch cohort of patients referred for
investigation of a possible bleeding disorder, the median BAT score was
4.0, while in the 2017 Vienna Bleeding Biobank study, it was 5.2,3 This

Haemophilia. 2022;28(Suppl. 4):119–124. wileyonlinelibrary.com/journal/hae © 2022 John Wiley & Sons Ltd. 119

120
does not necessarily mean that there is no real suspicion of a bleeding
disorder: a single very suspect bleeding event in a young patient with
no further challenges maybe a valid indication for haemostatic evalua-
tion. However, it could also indicate that the threshold for referral was
too low.
Overall, that is without exclusion based on the review of bleeding
symptoms, tertiary centres using advanced haemostatic testing reach
a final diagnosis of a defined bleeding disorder in 30%-50% of those
referred.2-4
In the absence of a diagnosis, no disease-specific treatment can be
prescribed. For mild bleeding disorders, this might be less of a problem
than expected. Two retrospective cohorts described the outcome of a
standard regime of tranexamic acid and/or desmopressin in patients
with unexplained bleeding. In the first, with 53 low-risk surgical pro-
cedures, 16 high risk and 13 deliveries, this was associated with the
absence of bleeding in 75/82.5 In the second cohort, 70 out of 78 pro-
cedures (including minor and major surgery, and deliveries) had good
haemostatic outcome.6 Of course, tranexamic acid is often also used
for minor interventions and menorrhagia in patients with a specific
diagnosis of a mild bleeding disorder.
Failing to identify the molecular cause of a heritable thrombocy-
topaenia often leads to a misdiagnosis of immune thrombocytopae-
nia and results in unnecessary immunosuppressive therapy or even
splenectomy. In the contemporary McMaster thrombocytopaenia reg-
ister, five out of 295 patients with an initial diagnosis of ITP were reclas-
sified as having inherited thrombocytopaenia during follow-up.7 This
demonstrates the importance of obtaining a family history and enquir-
ing after syndromic features prior to making a diagnosis of ITP. To date,
and reflecting the recent introduction of widespread genetic testing, no
systematically collected data are available on how often genetic diag-
nosis leads to a change in treatment plan.
When no specific diagnosis can be made, it is difficult to discuss
prognosis: How big is the risk that certain procedures are complicated
by bleeding? A recent study reported that, in 90 patients with bleed-
ing of unknown cause, higher bleeding scores were associated with an
increased risk of future bleeding.8 However, the size of the effect was
too small for useful prediction on an individual level. Uncertainty of
bleeding risk can lead to avoidance of procedures. For instance, neu-
raxial analgesia might be denied to women in childbirth. Lastly, a defini-
tive molecular diagnosis makes screening of family members more
accurate. This is especially important if family members have not had
haemostatic challenges and need to undergo major procedures.
In this review, we will introduce some hypothetical case scenarios,
explore the laboratory and genomic options for diagnosis and subse-
quently discuss how this might be applied in these cases.
2 CASE SCENARIOS
2.1 Case 1
Mr A, 56 years old, was diagnosed with mild haemophilia A in the
1990s. He has not been seen in the Haemophilia Treatment Centre
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MEIJER ET AL .
for at least 10 years, and no treatment with clotting factors or desmo-
pressin is documented. His daughter, who presented for preconception
advice, has a normal factor VIII level and no bleeding symptoms. She
undergoes genetic analysis specifically for haemophilia A, which does
not identify any potentially pathogenic variants in the F8 gene.
2.2 Case 2
Ms B, 18 years old, was referred from a community hospital with
mild thrombocytopaenia, diagnosed when she presented with bleed-
ing after dental extraction. She uses oral contraceptives for heavy men-
strual bleeding and tonsillectomy as a toddler was complicated by
bleeding immediately after the procedure. She is otherwise healthy.
Except for a possible mild bleeding tendency in her mother, there
was nothing remarkable in the family history. Platelet count was
93 × 109 /L, MPV reported as ‘not measurable’, with an excess of imma-
ture platelets. Light transmission aggregometry was confused by the
intermittent, ill-documented use of non-steroidal anti-inflammatory
drugs, but seemed decreased with adrenaline and collagen.
2.3 Case 3
Ms N, 26 years old, was referred for investigation of a bleeding his-
tory because she wished to start a family. Her major complaints were
severe menorrhagia that persisted despite the commencement of the
oral contraceptive pill and necessitated iron supplementation. In con-
junction with other episodes of unexplained bleeding, her BAT score
was 9. Her father had recurrent epistaxis, whereas her mother and sis-
ter had no bleeding symptoms. Previously, her bleeding time was pro-
longed and clotting screen revealed a prolonged PT and APTT. Fac-
tor assays showed an isolated mild factor II deficiency, and borderline
results in the von Willebrand factor assays.
3 PHENOTYPIC LABORATORY ANALYSIS
A thorough laboratory analysis to diagnose the patients’ anomaly
requires a detailed analysis of the haemostatic system (Figure 1). As
this is an interaction between vessel wall, platelets, coagulation and
fibrinolysis, an in-depth analysis can be laborious, time-consuming and
expensive.
Theoretically, the best approach is to have sensitive screening
assays for each compartment. Apart from vessel wall abnormali-
ties that cannot be covered with laboratory assays, easy-to-perform
screening assays only exist for platelet number and coagulation factor
deficiencies other than von Willebrand disease. Prothrombin time (PT),
activated partial thromboplastin time (APTT) and thrombin time (TT)
are typical coagulation screening assays.
A positive result in one of the screening assays streamlines identi-
fication of the patient’s anomaly as it directs confirmatory assays. The
analysis is hampered in cases where screening assays are completely

MEIJER ET AL .
F I G U R E 1 Flow chart of a laboratory assessment in a patient with
a suspected bleeding tendency. Green arrows indicate a negative
result, whereas a red arrow confirms an abnormality
normal, but the patient has an elevated BAT score or history suggestive
of a bleeding tendency. In these cases, several assays need to be per-
formed for abnormalities that are not adequately covered by screen-
ing assays. This might occur with mild disorders such as von Willebrand
disease or platelet dysfunction for which there is wide variation in the
assays used in specialist laboratories. Finally, if all screening and sec-
ondary assays are negative, further tests might include analysis of fibri-
nolysis (eg with the euglobulin clot lysis test, ECLT) and global assays of
haemostasis. The fibrinolysis panel can even be expanded by perform-
ing assays with plasma obtained before and during venous occlusion.
An important limitation of the current laboratory workflow is that
screening assays only analyse a small part of the haemostatic balance
(Figure 2). Prolonged clotting times generally indicate an abnormal-
ity in secondary haemostasis, and there is a lack of effective screening
assays for defects in primary haemostasis. Bleeding symptoms may be
due to a combination of defects in distinct haemostatic components.
In addition, confirmatory assays generally only focus on one parame-
ter. The so-called ‘global assays’, such as rotational viscoelastography,
thrombin generation, plasmin generation or a combination of both in
platelet-poor plasma, platelet-rich plasma or whole blood, may provide
more information as these assays are sensitive to multiple parts of the
haemostatic balance.
Despite the increasing use of global assays, the lack of standardiza-
tion and specificity limits utilization in diagnostic pathways. Neverthe-
less, it might be of interest to determine what the benefit would be
if these global assays are incorporated. Would these assays allow the
detection of more abnormalities compared with the current panel of
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121
F I G U R E 2 Potential assays to detect abnormalities in the
hemostatic pathway. The grey boxes represent assays in patients with
a suspected bleeding tendency. The red boxes represent assays in case
of thrombosis. The text in green describes global assays. Act, activity;
Ag, antigen; APTT, activated partial thromboplastin time; BT, bleeding
time; Fg, fibrinogen; PFA, platelet function analyser; Plt, platelet; PT,
prothrombin time; VWF, von Willebrand factor
assays, do they detect combinations of mild abnormalities and can they
be used to direct management? These are important questions to be
addressed in the future.
4 NEXT-GENERATION SEQUENCING (NGS) IN
THE INVESTIGATION OF RARE BLEEDING
DISORDERS
Understanding the molecular basis of a genetic disorder is essential for
accurate clinical diagnosis and development of disease-specific thera-
pies. This is very well demonstrated by the evolution of treatments for
haemophilia. In the last 3 decades, we have witnessed the widespread
use of recombinant factor concentrates, development of molecules
that bypass the deficient factor and the introduction of gene therapy
with the potential for a permanent cure. None of these treatments
would have been possible without the discovery and characterization
of the F8 gene.9 In fact, it was not gene discovery per se that was
important, rather it was that it led to routine use of genetic diagnosis
in the clinical assessment of haemophilia. The EAHAD database cur-
rently lists just over 3000 unique F8 variants from more than 10 000
haemophilia A cases (http://dbs.eahad.org/). It is the insights that we
have gained from the analysis of the effects of pathogenic and benign
variants that have provided the depth of molecular understanding nec-
essary for the breakthroughs listed above. As a direct result of the rou-
tine clinical use of genetic diagnosis, persons with severe haemophilia
A now have a variety of novel treatment options that offer a normal life
expectancy without severe bleeding-related morbidity.
Unfortunately, genetic diagnosis has not been so easy for bleeding
disorders other than haemophilia until now. There have been several
reasons for this. A few genes, such as VWF, are inherently difficult
to analyse due to structural complexities such as pseudogenes. Stan-
dard sequencing techniques based on the polymerase chain reaction
are costly and time-consuming when applied separately to genes

122
responsible for rare bleeding disorders. This is well demonstrated by
the fact that the EAHAD VWF database has approximately half the
number of unique variants listed compared with F8, despite von Wille-
brand disease being 10 times more common. Essentially, it has simply
been too expensive and laborious to support routine sequencing of
rare disorders using standard sequencing techniques that analyse part
of a single gene at a time.
The Human Genome Project completed in 2003 required the devel-
opment of techniques that allowed the quick analysis of vast sequences
of DNA in parallel.10 These techniques are collectively referred to as
‘high-throughput’ or ‘next-generation’ sequencing. They have provided
access to the whole genome, which in the case of humans is 3 billion
base pairs. Costs have plummeted and now we can analyse the whole
genome, and all of the 20 000 or so protein-coding genes that it con-
tains, for less than the price of a single gene analysis 10 years ago.
Alongside these new techniques, we have seen the development of
bioinformatic tools to process the vast amount of data generated. Fil-
tering of variants against the allele frequencies listed in large genomic
databases of apparently normal individuals, in silico analysis of their
effect on gene structure and protein function and use of algorithms to
collate this evidence are part of the several layers of analysis required
to determine whether a variant is pathogenic or not. However, filter-
ing using allele frequency can exclude some common pathogenic vari-
ants. This can be mitigated by the use of ‘whitelists’. These advances
had allowed several projects in different countries that have focused
on investigating the genetic basis of rare bleeding disorders.11-14 There
have been several benefits: patients with rare heritable bleeding dis-
orders can now receive a genetic diagnosis as easily as patients with
haemophilia, our understanding of the molecular basis of rare disease
and phenotypic effects is rapidly increasing and many new genes that
cause bleeding disorders have been discovered.
If we take the example of Hermansky-Pudlak syndrome, then prior
to the advent of NGS there were only two genes with a proven link to
the disease. Few patients could access genetic diagnosis and all were
considered to be at risk of serious sequelae such as pulmonary fibrosis
and granulomatous colitis. We now have at least 10 causative genes,
and only three are associated with pulmonary fibrosis. Increasing use
of genetic diagnosis has allowed lung screening to be restricted only to
those patients that need it.
Accurate genotyping of rare disorders has allowed better mapping
of non-haemostatic clinical features. For genes encoding transcription
factors such as RUNX1, ETV6 and GATA1, this has led to a better under-
standing of the risk of developing leukaemia.15 In other haematological
stem cell disorders, identification of the molecular aetiology has led to
the development of specific molecular therapies, such as the introduc-
tion of tyrosine kinase inhibitors following on from the characteriza-
tion of the BCR-ABL oncogene. At the moment, identification of these
variants does not improve clinical outcomes, but we can confidently
expect that specific molecular therapies will become available for those
at risk of malignancy due to the variants in the above genes.
In some cases, the accurate association of phenotype with geno-
type has resulted in a revaluation of the clinical features of well-
characterized conditions. May-Hegglin anomaly was considered to
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MEIJER ET AL .
be a relatively benign disorder characterized by thrombocytopaenia
and relatively little other pathology. It is now clear that the same
causative variants in MYH9 are also seen in the more serious conditions
Epstein, Fechtner or Sebastian syndrome in association with presenile
cataracts, hearing loss and renal failure.16 These eponymous names
implying distinct conditions no longer reflect the genotype-phenotype
relationship with these genes. The single overarching term, MYH9-
related thrombocytopaenia, better reflects our current understanding
that the spectrum of disease encompasses all these clinical features.
While NGS has enabled access to genes for which testing was not
previously feasible in a clinical setting, the efficiency of simultaneous
multigene testing does come with some disadvantages. Incidental find-
ings are an obvious example. Currently, the available platforms can be
divided into those that offer whole-genome testing and those that offer
gene panels which group together all the genes that are of relevance for
a particular field. The genomics scientific subcommittee of the Inter-
national Society on Thrombosis and Haemostasis maintains a list of
genes that are considered to be proven to cause haemostatic disease
at https://www.isth.org/page/GinTh_GeneLists. As of February 2020,
there are 101 genes in this list of which 22 are coagulation factor genes,
11 are thrombotic and 68 cause platelet abnormalities. A gene panel
for clinical use in the field of haemostasis would be expected to contain
all these genes with the option for revision as new genes are added to
this list. The likelihood of an incidental finding is relatively low with a
gene panel. This was demonstrated in the ThromboGenomics project
which investigated nearly 2400 patients with haemostatic disease and
resulted in only 6 incidental findings that were considered reportable
under current guidance.11 With whole-genome analysis that poten-
tially gives access to an estimated 20 000 genes, the potential for inci-
dental findings is obviously much greater. The handling of potentially
pathogenic variants in genes unrelated to the field of interest requires
extensive interaction between different clinical disciplines and is dif-
ficult to implement in current clinical services. It is unclear whether
patients receive a net benefit from these findings, and this raises impor-
tant ethical considerations.17 For the moment, clinical services are
largely based on gene panels with whole-genome analysis mostly used
in research.
Finally, it is important to be aware of the limitations of any diagnos-
tic tool. The chance of finding a potentially pathogenic variant depends
on the type of haemostatic disorder under investigation. In the Throm-
boGenomics study, 67% of patients with mostly rare coagulation fac-
tor deficiencies had a pathogenic or potentially pathogenic variant. For
patients with heritable disorders of platelet number and/or function,
the diagnosis rate was rather lower at 38% because the genetic basis of
these disorders is much less well-characterized. Often, there is simply
insufficient knowledge of the effects of variation in a specific gene to be
confident of predicting pathogenicity. Co-segregation studies may help
in large pedigrees with clearly defined laboratory assays for a specific
phenotype. Where this is not feasible, it is expected that the accumu-
lation of ‘big data’ through international collaborative databases will
facilitate curation. NGS remains primarily a sequencing tool that has
a very high accuracy for single nucleotide changes. Copy number vari-
ants may also be detected with comparative analysis of read depth,

MEIJER ET AL .
but complex rearrangements are not detected. An important exam-
ple is the intron 22 inversion in F8, which is the commonest cause of
haemophilia A, and is undetectable by current NGS techniques. We also
have to be mindful that pseudogenes and repetitive elements can lead
to misleading results and coverage of the non-coding space is of inad-
equate quality for many genes. All these factors need to be considered
when interpreting negative reports.
5 DISCUSSION
5.1 Case 1, continued
Current genetic testing strategies fail to identify the causative vari-
ant in about 5% of cases of non-severe haemophilia A. Non-paternity,
mosaicism and an alternative diagnosis are also possible explanations
for the absence of a variant in a daughter expected to be an obli-
gate carrier. In this case, genetic testing of the index revealed homozy-
gosity for a variant previously reported in two families with type 2N
von Willebrand disease. Subsequently, further genetic analysis on his
daughter showed her to be heterozygous for the same variant.
5.2 Case 2, continued
In Ms B, the application of an NGS panel focused on primary haemosta-
sis showed heterozygosity for a previously reported variant in the
MYH9 gene. On further questioning, the family history was negative for
renal disease, cataract or hearing loss. Additional testing revealed pro-
teinuria with intact renal function in the patient, and she was referred
to a nephrologist.
5.3 Case 3, continued
Genomic panel testing revealed heterozygosity for a nonsense vari-
ant in VWF that was classed as pathogenic, and heterozygosity for a
likely pathogenic variant in F2. The introduction of a premature stop
codon in one VWF allele would explain the borderline levels of this fac-
tor and would be expected to result in type 3 disease in the homozy-
gous state. Similarly, the missense variant in one F2 allele is consistent
with the mild reduction in factor II levels. In isolation, neither of these
variants would be expected to result in significant bleeding symptoms,
but it may be that the combination of both could result in the pheno-
type described.
For many patients, the developments discussed in this paper have
led to improved diagnostics, within some cases access to specific ther-
apy or diagnostic possibilities for family members. A comprehensive
list of outcomes after extensive investigations is given in the EHA Con-
sensus Report.1 In our first case, the correct diagnosis of type 2N von
Willebrand disease led to a modified treatment plan for the index case
and appropriate counselling of his daughter. As the cost of genomic
testing falls and access increases, it has been suggested that it could
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123
be utilized earlier in the diagnostic pathway. In contrast with assays
such as platelet aggregometry, which require the patient to attend a
specialist centre for a fresh blood sample, samples for genomic testing
can be taken remotely, perhaps even non-invasively with saliva sam-
ples, and sent by ordinary post to the testing laboratory. However,
except for a few well-characterized variants that have been reported
often enough that pathogenicity classification is automatic, interpre-
tation still requires correlation with laboratory assays to establish
the genotype-phenotype relationship. The laboratory assays described
above are unlikely to become redundant any time soon.
Genetic diagnosis can also have drawbacks, especially in the diag-
nosis of platelet disorders with unanticipated clinical features. This
is illustrated by the second case, where the patient did not make an
explicit choice to know about her increased risk of renal disease. How-
ever, for MYH9 disease, the diagnosis gives opportunities for preventa-
tive action (ie follow-up of renal function, initiation of angiotensin inhi-
bition). This is not always the case.
Inherited platelet disorders can be subdivided into three groups,
based on their additional features: ‘affecting only platelets’, ‘with
a syndromic phenotype’ and ‘with increased risk of haematological
malignancies’.18 For this third group, the consequences of genetic test-
ing can be profound.19 It would be desirable to allow patients to make
an informed choice as to which genes are tested, thereby controlling
to some extent the possibility of unexpected findings. However, this
requires in-depth knowledge of testing strategy at the time of the
request and may not be possible depending on how the NGS platform is
set-up. This has wider ethical implications when the rights and wishes
of relatives are considered. Testing of multiple members of a pedi-
gree can infer genotype in untested relatives whether or not they have
agreed to testing. The ISTH scientific subcommittee for genomics in
thrombosis is currently preparing guidance on the ethical implications
of genomic testing for platelet disorders.
In conclusion, NGS has greatly improved our ability to provide
a definitive diagnosis in patients with a heritable platelet disor-
der. In patients with other rare bleeding disorders that were previ-
ously diagnosed phenotypically (ie coagulation deficiencies other than
haemophilia A or haemophilia B), advanced genetic testing provides
confirmation, better understanding of the molecular pathology and the
opportunity for prenatal testing. Additionally, NGS may streamline the
diagnostic process, replacing part of the traditional haemostatic work-
up.
ACKNOWLEDGEMENTS
KG was a chief investigator in the ThromboGenomics study. WvH is the
chief scientific officer of Enzyre.
DISCLOSURES
The authors stated that they had no interests which might be perceived
as posing a conflict or bias.
ORCID
Karina Meijer https://orcid.org/0000-0001-9447-0465
Keith Gomez https://orcid.org/0000-0002-8934-0700

124
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How to cite this article: Meijer K, van Heerde W, Gomez K.
Diagnosis of rare bleeding disorders. Haemophilia.
2022;28(Suppl. 4):119–124.
https://doi.org/10.1111/hae.14561