Rare Bleeding Disorders in the Netherlands
Conditions: Rare Bleeding Disorders
Sponsor: Radboud University Medical Center

NL61027.091.17 / National study of rare bleeding disorders in the Netherlands 
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Corresponding author:  
Dr. Marten R. Nijziel MD PhD 
Radboud university medical center, Department of Haematology, route 476 
P.O. Box 9101  
6500 HB Nijmegen  
The Netherlands 
Ph: +31 (0)24 3668286 
F: +31 (0)24 3542080 
E: marten.nijziel@radboudumc.nl 
Concept version 3.0, 16-8-2017  
 

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PROTOCOL TITLE ‘Rare Bleeding Disorders in the Netherlands (RBIN)’ 
 
Protocol ID 
HEMSTOL47 
Short title 
Rare bleeding disorders in the Netherlands 
Version 
2.0 
Date 
July 5, 2017 
Coordinating investigator/ 
project leader 
 
Dr. Marten R. Nijziel MD PhD 
Radboud university medical center, Department of 
Haematology, route 476 
P.O. Box 9101 
6500 HB Nijmegen  
The Netherlands 
P: +31 (0)24 3668286 
F: +31 (0)24 3542080 
E: marten.nijziel@radboudumc.nl 
Principal investigator(s) (in Dutch: 
hoofdonderzoeker/ uitvoerder) 
 
Multicenter study, participating sites and principal 
investigators: 
Academical Medical Center Amsterdam, Dr. Marjolein 
Peters MD PhD 
University Medical Center Groningen, Prof. Dr. Karina 
Meijer MD PhD 
Leiden University Medical Center, Dr. Felix J.M. van 
der Meer MD PhD 
Erasmus MC, Dr. Marjon Cnossen MD PhD 
UMCU, Prof. dr. Roger Schutgens MD PhD 
Máxima Medisch Centrum Eindhoven/Veldhoven, Dr. 
Laurens Nieuwenhuizen MD PhD 
Maastricht UMC+, Dr. Erik Beckers MD PhD 

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Investigators 
Drs. Joline Saes MD 
Dr. Saskia Schols MD PhD 
Dr. Britta Laros-van Gorkom MD PhD 
Drs. Yolba Smit MD 
Adinda Diekstra 
Ton de Haan MSc 
Dr. Sonja de Munnik MD PhD 
Dr. Paul Brons MD PhD 
Sponsor (in Dutch: 
verrichter/opdrachtgever) 
Research center Haemostasis Radboudumc 
Nijmegen   
Subsidising party 
Radboudumc 
Independent expert (s) 
Adults: Mevr. Dr. E.M. Klappe MD PhD, specialist in 
internal medicine, department Algemene Interne 
Geneeskunde, Radboudumc (024 – 3616504) 
 
Children: Dr. M. Schreuder MD PhD, paediatrician – 
nephrologist, department of paediatrics, Radboudumc 
(024 – 3655700) 
Laboratory sites 
Dr. Waander van Heerde PhD 
Laboratory Hematology 
Radboud university medical center 
 
 
 
 
 

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PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Acting Head of Department: 
 Dr. Michel Schaap MD PhD 
 
 
 
Coordinating Investigator 
Dr. Marten Nijziel MD PhD 
 
 
 
 

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TABLE OF CONTENTS 
TABLE OF CONTENTS ........................................................................................................ 5 
LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS ................................................ 8 
SUMMARY ...........................................................................................................................10 
1. 
BACKGROUND.............................................................................................................11 
1.1. 
Epidemiology of the rare bleeding disorders ...........................................................12 
1.2. 
Clinical manifestations ............................................................................................12 
1.3. 
Health-related quality of life ....................................................................................16 
1.4. 
Laboratory ..............................................................................................................16 
Screening assays ..........................................................................................................16 
Confirmation assays ......................................................................................................17 
Global assays ................................................................................................................17 
1.5. 
Genetics .................................................................................................................17 
1.6. 
Management ..........................................................................................................18 
1.7. 
Ageing and coagulation ..........................................................................................18 
1.8. 
Arteriothrombotic events and bleeding disorders ....................................................18 
2. 
RATIONALE ..................................................................................................................20 
3. 
OBJECTIVES ................................................................................................................22 
4. 
STUDY DESIGN ...........................................................................................................23 
5. 
STUDY POPULATION ..................................................................................................24 
5.1. 
Population (base) ...................................................................................................24 
Characteristics of the Dutch source population ..............................................................24 
Willingness of patients to participate ..............................................................................25 
5.2. 
Inclusion criteria .....................................................................................................25 
5.3. 
Exclusion criteria ....................................................................................................26 

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6. 
METHODS ....................................................................................................................27 
6.1. 
Study procedures ...................................................................................................27 
6.2. 
Data collection ........................................................................................................28 
Blood sampling ..............................................................................................................29 
Saliva sampling .............................................................................................................29 
Electronic patient records ..............................................................................................30 
Whole exome sequencing .............................................................................................30 
6.3. 
Withdrawal of individual subjects ............................................................................30 
6.4. 
Replacement of individual subjects after withdrawal ...............................................30 
6.5. 
Follow-up of subjects withdrawn from treatment .....................................................30 
6.6. 
Premature termination of the study .........................................................................30 
7. 
STATISTICAL ANALYSIS .............................................................................................31 
7.1. 
Primary objectives ..................................................................................................31 
7.2. 
Secondary objectives .............................................................................................32 
8. 
ETHICAL CONSIDERATIONS ......................................................................................34 
8.1. 
Regulation statement .............................................................................................34 
8.2. 
Recruitment and consent........................................................................................34 
8.3. 
Objection by minors or incapacitated subjects ........................................................34 
8.4. 
Benefits and risks assessment, group relatedness .................................................34 
8.5. 
Compensation for injury .........................................................................................34 
8.6. 
Incentives ...............................................................................................................35 
9. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .............................36 
9.1. 
Handling and storage of data and documents ........................................................36 
9.2. 
Monitoring and Quality Assurance ..........................................................................36 
9.3. 
Amendments ..........................................................................................................36 

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9.4. 
Annual progress report ...........................................................................................36 
9.5. 
Temporary halt and (prematurely) end of study report ............................................36 
9.6. 
Public disclosure and publication policy ..................................................................37 
10. 
REFERENCES ..........................................................................................................38 
Appendix 1: Patient information letter and informed consent form ........................................41 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
 
ALAT 
Alanine amino transferase 
ANOVA 
Analysis of Variance 
APTT 
Activated Partial Thromboplastin Time 
CI 
Confidence Interval 
F 
Factor 
GCP 
Good Clinical Practice 
HIN 
Haemophilia in the Netherlands 
ICH 
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
ISTH(SSC)-
BAT 
International Society on Thrombosis and Haemostasis (Scientific and 
Standardization Committee)- Bleeding Assessment Tool 
METC  
Medical research ethics committee (in Dutch: medisch ethische toetsings 
commissie) 
NHA 
Novel Haemostasis Assay 
NVHP 
Netherlands Haemophilia Society; (in Dutch: Nederlandse Vereniging van 
Hemofilie patiënten) 
PAI 
Plasminogen Activator Inhibitor 
PFA 
Pro-RBDD 
Platelet Function Analyser 
Prospective Rare Bleeding Disorder Data register 
PT 
Prothrombin Time 
TAFI 
Thrombin Activatable Fibrinolysis Inhibitor 
TiN 
Thrombopathy in the Netherlands 
TPA 
Tissue-type Plasminogen Activity 
TT 
Thrombin Time  

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TSH 
Thyroid Stimulating Hormone 
VWF 
Von Willebrand Factor 
Wbp 
Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens) 
WES 
Whole Exome Sequencing 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 

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SUMMARY 
Rationale: Rare bleeding disorders (deficiency of fibrinogen, factor II, V, V&VIII, VII, X, XI, 
XIII, α2-antiplasmin or plasminogen activator inhibitor 1) are not well defined with respect to 
their clinical phenotype, laboratory phenotype en genotype. At present, little is known about 
their clinical presentation, bleeding scores, bleeding episodes, health-related quality of life, 
laboratory parameters, genetics and current treatment. There are large differences in 
bleeding tendency and weak correlations with the level of factor deficiencies. Therefore, it is 
essential to perform thorough research in patients with rare bleeding disorders and perform 
laboratory and genetic tests, to seek explanations for the variety in clinical phenotype. 
Objective: The purpose of the RBiN study is to describe the epidemiology, bleeding 
tendency, laboratory parameters, quality of life and genetics of all known patients in the 
Netherlands with rare bleeding disorders. In addition, the study aims to examine the 
relationship between clinical phenotype, laboratory phenotype and genotype.  
Study design: cross-sectional multicenter observational study. 
Study population: all patients registered in Dutch Haemophilia Treatment Centers with 
known disorders of the coagulation factors fibrinogen, factor II, V, V & VIII, VII, X, XI, XIII, α2-
antiplasmin and plasminogen activator inhibitor type 1, aged 1 years and older. 
Main study parameters/endpoints:  
Description of the clinical phenotype, laboratory phenotype, genotype and quality of life. 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: participating patients will be invited for one visit to their treatment center 
in order to draw blood, take a saliva sample and perform questionnaires. This will take 
approximately 40 to 120 minutes. Since the population of patients with RBDs is very small it 
is important to include all patients, also minors (children <18 years), in the study (around one 
third of known patients are minors). Therefore, this study may be regarded as group-related. 
The risk associated with participation is negligible. 

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1. BACKGROUND  
Research of inherited bleeding disorders traditionally focuses on haemophilia because of the 
relatively ‘common’ presentation combined with a dramatic clinical picture. Haemophilia A - 
deficiency of coagulation factor (F) VIII - , and haemophilia B - deficiency of FIX – occur at an 
incidence of 1 in 10,000 and 1 in 60,000 live births, respectively [1]. In contrast, the rare 
bleeding disorders affecting coagulation factors fibrinogen, factor II, V, V&VIII, VII, X, XI and 
XIII, α2-antiplasmin and plasminogen activator inhibitor type 1 (PAI-1) are 10 to 200 times 
less frequent than haemophilia, with a general population prevalence estimated at 1:500,000 
up to 1 per two million (Table 1Table 1) [1, 2]. Worldwide 16,000 patients have been 
registered though this is likely a serious underestimation of the actual patient number [3]. 
Their extreme rarity, clinical heterogeneity, difficulty in recognising affected patients, limiting 
laboratory assays and challenges in collecting longitudinal data have led to a gap in 
knowledge on all aspects of these rare disorders, and possibly to an underreporting of less 
severe cases. It is therefore imperative to bundle research efforts at a national (or even 
international) level to gain insight, as few centers have the possibility to follow and manage a 
consistent number of patients. 
Table 1 General population prevalence of homozygous coagulation factor disorders 
Disorder 
Full name and variants (also known as) 
Estimated general 
population prevalence 
homozygosity 
Coagulation factor 
disorders 
 
 
- 
I 
Fibrinogen disorders 
 
Afibrinogenemia  
 
Hypofibrinogenemia 
 
Dysfibrinogenemia 
1:1 million [1, 2] 
- 
II 
 
Prothrombin deficiency 
(hypoprothrombinemia) 
 
Dysprothrombinemia 
1:2 million [1, 2] 
- 
III 
Platelet tissue factor: deficiency assumed to be incompatible with life [4] 
- 
IV 
Calcium: no longer considered a ‘mere’ coagulation factor 
- 
V 
Factor V deficiency (proaccelerin deficiency, labile 
factor deficiency, Owren’s disease, parahemophilia) 
1:1 million [1, 2] 
- 
V & VIII 
Combined FV & FVIII deficiency 
1:1-2 million [2] 
- 
VI 
Activated form of FV, no longer recognised as a coagulation factor on its own 
- 
VII 
Factor VII deficiency (proconvertin deficiency, 
Alexander’s disease) 
1:500,000 [1, 2] 
- 
VIII 
Haemophilia A (classic haemophilia) 
1:10,000 
- 
IX 
Haemophilia B 
1:60,000 
- 
X 
Factor X deficiency (thrombokinase deficiency 
1:500,000-1 million [1, 2, 5] 

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,Stuart-Prower factor deficiency) 
- 
XI 
Factor XI deficiency (plasma thromboplastin 
antecedent deficiency, Rosenthal syndrome, 
haemophilia C) 
1:1 million [1, 2] 
- 
XII 
Factor XII deficiency (Hageman factor deficiency; not associated with bleeding 
tendency) 
- 
XIII 
Factor XIII deficiency (fibrin stabilising factor 
deficiency) 
1:1-2 million [1, 2] 
Fibrinolysis disorders 
 
 
- 
α2-Antiplasmin  α2-Antiplasmin deficiency  (Miyasato disease) 
Unknown, 
40 cases reported 
worldwide [6, 7] 
- 
Plasminogen 
activator inhibitor 
type 1 
Plasminogen activator inhibitor type 1 deficiency 
Unknown, few cases 
reported worldwide [8, 9] 
 
1.1. Epidemiology of the rare bleeding disorders 
Rare bleeding disorders are scarce. General population prevalence for homozygosity is 
estimated at 1:500,000 for factor VII deficiency up to 1 per million or 1 per two million for the 
disorders of fibrinogen, and the factor II, V, V&VIII, X, XI, and XIII deficiencies (Table 1Table 
1) [1, 2]. These estimates originate from case descriptions, case series or at best registry 
studies, undertaken several decades ago. At present, these estimated prevalences are the 
best we have, and they are generally accepted. In general, data from registries and case 
series are prone to underestimating the true population prevalence because of problems with 
capturing the entire population and underreporting of less severe cases [3]. Reported 
prevalences of patients with clinically significant deficiencies (factor levels of 10% or less) 
registered in comprehensive registries in Iran, Italy and the United Kingdom are of the same 
magnitude as the population prevalence estimates [10].  
1.2. Clinical manifestations 
Patients affected by rare bleeding disorders have a wide spectrum of clinical presentations 
that varies from a mild or moderate bleeding tendency to potentially serious or life-
threatening haemorrhages [11].  
Occasionally, the interplay with other inherited coagulation disorders such as platelet defects, 
von Willebrand disease and other coagulation factor deficiencies may give a more severe 
bleeding tendency [12-18].  
There is a heterogeneous association between coagulation factor activity level and clinical 
bleeding severity in the different rare bleeding disorders. The coagulation factor activity 
levels that are necessary for patients to remain asymptomatic are [19]: 

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 
Fibrinogen: > 100 mg/dL;  
 
FV: 12%;  
 
FV & VIII: 43%;  
 
FVII: 25%;  
 
FX: 56%;  
 
FXI: 26%; 
 
FXIII: 31% 
 
Coagulation factor activity levels that corresponded with spontaneous major bleeding were 
[19]: 
 
Fibrinogen: undetectable levels;  
 
FV: undetectable levels; 
 
FV & VIII: 15%; 
 
FVII: 8%; 
 
FX: 10%; 
 
FXI: 25%; 
 
FXIII: undetectable 
For the fibrinolysis disorders, there are no data available on the factor activity levels 
necessary to remain asymptomatic. Available data on patients with rare bleeding disorders 
are based on clinical records, which will predominantly include patients presenting with more 
severe bleeding symptoms. This generates a high risk of ascertainment bias, with a possible 
overestimation of the severity of bleeding in both homozygous and heterozygous individuals 
at group level. 
Fibrinogen disorders 
Afibrinogenemia (defined as fibrinogen <200 mg/L) is inherited in an autosomal recessive 
pattern. Patients with congenital afibrinogenemia suffer from a lifelong severe bleeding, with 
haematomas, haematemesis, melaena, or umbilical cord bleeding manifesting in the 
newborn period in severe cases [20]. Internal bleeding and spontaneous splenic rupture are 
common features, as is severe bleeding after minor trauma, easy bruising and gingival 
haemorrhage. In a study from 1971 that described all known case-reports of afibrinogenemia 
up to that time (n=23) 52% of patients reported umbilical bleeding, 70% severe bleeding after 
minor traumata and 13% central nervous system bleeding [21]. Arterial and venous 
thromboembolic complications were also recorded in some cases, as well as recurrent 
miscarriage [22]. 
Dysfibrinogenemia is usually asymptomatic but may present as a (severe) bleeding tendency 
[20]. It inherits either autosomal recessively or dominantly. Thrombosis is a reported 
complication of dysfibrinogenemia in 10 to 30% of patients and there may be impaired wound 

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healing [20]. Recurrent miscarriage is seen in both afibrinogenemia and dysfibrinogenemia 
[20]. 
Hypofibrinogenemia, which is inherited in an autosomal dominant pattern, is defined as 
fibrinogen <500mg/L. It also presents with a bleeding tendency. In an Iranian series of 60 
patients, all with a fibrinogen level below 1 g/L, 75% reported cord bleeding, 70% oral cavity 
or nose bleeding, 50% joint bleeding or uterus bleeding, 40% postoperative- or post-partum 
bleeding, and 5% central nervous system bleeding [23].  
FII deficiency 
FII deficiency is an autosomal recessive disorder. Severe FII deficiency can lead to bleeding 
of the umbilical cord, joint, muscle and mucosal bleeding, central nervous system bleeding 
and bleeding after invasive procedures. In a case series of 13 patients 15% of patients had 
had an umbilical cord bleeding and 15% a central nervous system bleeding [24]. In 39 severe 
cases reported in the literature  intracerebral and gastrointestinal bleeding was seen in 12% 
of patients, and haemarthrosis in 42%[25]. Spontaneous haematomas/bruising were seen in 
60% of patients, bleeding after tooth extractions in 36%, and 20% of females reported 
menorrhagia.  
FV deficiency 
FV deficiency is an autosomal recessive disorder. The bleeding tendency in FV deficiency is 
variable and does not correlate well with the level of FV, especially in the low (<5%) range 
[22]. The explanation for this heterogeneity is unknown, but compensation by platelet FV 
may play a role [26]. According to Iranian and North American registries the most common 
symptoms associated with FV deficiency were bleeding from mucous membranes (e.g. 
epistaxis, menorrhagia in females) and post-traumatic bleeding following surgery or delivery, 
which occurred in approximately half of patients[27]. Haemarthroses and muscle 
haematomas were present in around one quarter of patients, and severe bleeding 
manifestations (e.g. intracranial or gastro-intestinal haemorrhages) were rare and confined to 
patients with undetectable FV levels. Despite this rather benign clinical picture there have 
been several reports in the literature on patients presenting with life-threatening neonatal or 
perinatal intracranial haematoma’s [27]. 
FV & FVIII deficiency 
Combined FV & FVIII deficiency is an autosomal recessive disorder. Individuals with a FV 
and FVIII deficiency do not have a more severe bleeding tendency than individuals with 
either a FV or a FVIII deficiency [28]. Those affected usually have a mild to moderate 
bleeding tendency, with mucous membrane bleeding like epistaxis (19 to 77%), gum 
bleeding (49 to 64%), menorrhagia (66 to 100%) and post-traumatic/post-surgery (62 to 
85%) as the most common manifestations [29]. Joint and muscle bleedings were reported in 
25% and 7% of 27 patients in one series, but it was not reported whether these bleedings 
were spontaneous [23]. In the same patient series 22% reported cord bleeding and 4% 
central nervous system bleeding.  

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FVII deficiency 
FVII deficiency is an autosomal recessive disorder. The clinical manifestation is 
heterogeneous. There is a weak correlation between the level of FVII and bleeding tendency. 
Mild disease will lead to easy bruising, bleeding from mucous membranes or menorrhagia 
[5]. Severe and homozygous FVII deficiency may lead to haemarthrosis, cerebral 
haemorrhage and catastrophic haemorrhage after parturition, surgery or injury [5, 30]. In a 
large series of 73 homozygous individuals intracranial haemorrhage (2%), gastrointestinal 
bleeding (17%), haemarthrosis (13%), epistaxis (58%), gum bleeding (38%), easy bruising 
(37%), haematoma (15%), hematuria (10%) and menorrhagia (73%) were described [31]. 
Interestingly, 19% of heterozygous individuals reported spontaneous bleeding, though 
severe spontaneous haemorrhages (intracranial and gastrointestinal) were not reported in 
this series [31]. In contrast, in a series of 49 patients with <10% FVII 17% reported central 
nervous system bleeding and 14% gastrointestinal bleeding [23].  
FX deficiency 
FX deficiency is an autosomal recessive disorder. The risk of bleeding correlates well with 
coagulation factor activity level [19]. Severe bleeding occurs in patients with factor levels 
below 10%, and includes umbilical cord, intracranial, joint and muscle bleeding and 
excessive bleeding after surgery or trauma. Heterozygous individuals can have a bleeding 
tendency. [32].  
FXI deficiency 
FXI deficiency can be inherited autosomal recessive and autosomal dominant[33]. It is the 
mildest of the rare bleeding disorders, with bruising, epistaxis and menorrhagia as the most 
common manifestations [5, 14]. The correlation between coagulation factor activity and 
clinical bleeding is poor [19]. This may (in part) be explained by the coexistence of other 
coagulation disorders. In some patients with severe FXI deficiency a combined FXI and von 
Willebrand disease has been reported, leading to a more severe bleeding tendency [16]. 
Excessive bleeding has also been reported in heterozygous individuals. A single study from 
the United Kingdom showed that 48% of heterozygous individuals had a bleeding tendency, 
[34]. Comparison of histories between partially deficient and non-deficient individuals 
demonstrated a higher prevalence of menstrual problems (41% vs. 18%), an increase in 
significant bruising (38% vs. 14%), an increased likelihood of excessive bleeding after dental 
extractions (51% vs. 9%), and an increased number of blood transfusions after interventions 
(56% vs. 0%). Bleeding in heterozygous individuals has also been described to be related to 
genetically compound disease, other factor deficiencies or defects, or platelet dysfunction in 
some cases [12-18].  
FXIII deficiency 
FXIII deficiency is an autosomal recessive disorder. This deficiency has serious clinical 
consequences. Of 112 cases reported in the literature from 1978 23 individuals (21%) had 
died of bleeding, and notably 22 of these were relatives of established cases, for whom the 
diagnosis was highly likely but could not be proven [35]. Umbilical cord bleeding was seen in 

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73-80% of cases and may be delayed up to 19 days after birth; surgical bleeding in 84%; 
superficial bruising in 58-60%; subcutaneous haematomas in 55-58%; intracranial bleeding 
in 25-30%; mouth and gum bleeding 30-48%; muscle bleeding 27%; and joint bleeding in 24-
55% of reported cases [35, 36]. Habitual abortions (50% of women) and menorrhagia (35% 
of women) are also frequent. Women with a complete deficiency are unable to carry a 
pregnancy to term [36, 37]. Impaired wound healing is another symptom patients may 
experience [37]. It is currently not clear if people with a heterozygous factor XIII deficiency 
have an increased bleeding risk[38]. 
 
Alpha-2-antiplasmin deficiency 
Alpha-2-antiplasmin deficiency is an autosomal recessive disorder. Most patients with alpha-
2-antiplasmin deficiency experience severe bleeding appearing during childhood. Umbilical 
cord bleeding has been described, as well as intramedullary haemorrhage into the diaphyses 
of long bones. Heterozygous individuals can be asymptomatic or have milder symptoms, 
mostly occurring after trauma, surgery or dental extractions. With age the plasma levels 
decrease which can lead to increase in symptoms [6, 7]. 
Plasminogen activator inhibitor deficiency 
PAI-1 deficiency is an autosomal recessive disorder. The bleeding symptoms in PAI-1 
deficiency are usually mild to moderate. Bleeding after surgery is a common complication as 
well as abnormal bleeding after dental procedures. Most bleeding episodes are post-
traumatic. Epistaxis has also been described as well as menorrhagia, sometimes leading to 
iron deficiency anaemia or even the need for transfusion [8, 9]. 
1.3. Health-related quality of life 
Health-related quality of life has not been evaluated in patients with rare bleeding disorders.  
1.4. Laboratory 
The laboratory diagnosis of rare bleeding disorders consists of screening assays, 
confirmation assays, and global assays. Though characterization of the bleeding disorder is 
possible through this diagnostic cascade, either or not in combination with genotypic 
confirmation, there is no test available that sufficiently predicts bleeding phenotype. In 
addition, all currently available laboratory tests necessitate venepuncture. Non-invasive 
and/or home-based assays do not exist as yet. 
Screening assays 
If a bleeding disorder is suspected, clinicians will request coagulation tests and tests to 
exclude disorders that influence coagulation, such as thyroid disease or liver disease. A 
routine screening assay generally  includes the following tests: activated partial 
thromboplastin time (APTT), prothrombin time (PT) and fibrinogen activity or thrombin time 
(TT); platelet function analyzer (PFA), von Willebrand factor activity and antigen, FVIII, 

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fibrinogen, platelet count, haemoglobin, leucocytes & differentiation, peripheral blood smear, 
creatinine, ALAT (alanineaminotransferase) and TSH (thyroid stimulating hormone). A 
prolonged aPTT or PT without another deviating test may indicate a rare bleeding disorder, if 
acquired diseases and anticoagulant use have been ruled out. A prolonged PT indicates a 
deficiency of FVII, FX, FV, FII, or fibrinogen. A prolonged aPTT indicates a deficiency of 
either prekallikrein, fibrinogen, FII, FV, FVIII, FIX, FXI of FXII. A prolonged aPTT and PT 
suggests that the abnormality is due to a deficiency in FX, FV, FII or fibrinogen.  
Confirmation assays 
Specific factor assays for each different coagulation factor confirm the specific rare bleeding 
disorder, and are necessary to give a rough indication of the level of deficiency (severe, 
moderate, mild). Functional assays are indicated if antigen levels are normal, slightly 
reduced or increased because they can confirm qualitative factor disorders. Genetic testing 
is not routine practice, as it does not have direct clinical implications, but may be indicated in 
case of preconception counselling. 
Global assays 
The Nijmegen Haemostasis Assay (NHA) measures thrombin and plasmin generation in a 
single well and allows the detection of coagulation, fibrinolysis and their interplay in a single 
assay [39]. Preliminary data of 41 patients suggest that the NHA can distinguish fibrinogen 
disorders and FII, FV, FVII, FX and FXIII deficiencies [40].  
1.5. Genetics 
In general, mutations in genes that encode for the corresponding coagulation factor are the 
cause of the bleeding disorder, and mutations are almost always unique to single kindred 
(Table 2Table 2).  The combined deficiency of FV & FVIII is caused by mutations in genes 
encoding for proteins involved in intracellular transport [41]. Deficiencies in the vitamin K 
dependent coagulation proteins (FII, FVII, FIX and FX) can be caused by mutations in genes 
encoding enzymes involved in post-translational modification and in vitamin K metabolism 
[42].  
In approximately 10% to 20% of patients, no putative mutation is found with conventional 
genetic techniques that scan the ‘usual suspect’ genes. Cases were no putative mutation has 
been found may be due to defects in non-coding regions or in genes coding for regulators of 
intracellular transport and posttranslational modifications of coagulation factors. 
Table 2 General genetic features of the rare bleeding disorders [10, 23] 
Disorder factor 
Gene on chromosome 
Fibrinogen 
4 
II 
11 
V 
1 
V & VIII 
18 

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VII 
13 
X 
13 
XI 
4 
XIII 
A subunit: 6 
B subunit: 1 
PAI-1 
7 
Alpha-2-antiplasmin 
17 
 
1.6. Management  
Many patients and bleeding episodes will not require therapy: in a North-American registry 
study in 294 patients with a rare bleeding disorder half of all bleeding episodes did not 
require therapy [11]. On the other side of the treatment spectrum are patients with severe FX 
and FXIII deficiency, who may require lifelong prophylaxis treatment. If treatment is needed, 
the optimal treatment is often unknown as high-quality evidence is lacking. Both factor 
replacement therapy and non-transfusional treatment are available at present. The backbone 
of treatment is factor replacement, either in the form of recombinant factor, factor 
concentrate, prothrombin complex concentrate or plasma products (in order of preference 
and depending on availability). Non-transfusional treatment includes antifibrinolytic agents 
(tranexamic acid or ε-aminocaproic acid), which can be used as a supplement to factor 
replacement in major bleedings, and instead of replacement therapy in minor bleeding. 
Treatment-related complications have been reported and include viral seroconversion, 
anaemia, allergic reactions and venous access device-related events [11]. Cases of 
inhibitors to FV and FXI have been reported.  
1.7. Ageing and coagulation  
Aging has a distinct effect on coagulation in healthy individuals, with progressive laboratory 
evidence of increased coagulation activity as individuals age [43]. The plasma concentrations 
of fibrinogen, factor VII, factor VIII, von Willebrand factor (VWF), factor IX, factor XII, high 
molecular-weight kininogen, and prekallikrein increase with progressing age in healthy 
humans [44]. In centenarians, higher levels of fibrinogen, FII and FX have been found, 
compared to younger individuals, whereas levels of FVII, α2-antiplasmin and PAI did not 
differ between age groups [45]. It is unknown whether these changes in factor concentrations 
occur in patients with RBD and if they influence clinical phenotype. In von Willebrand disease 
age-dependent laboratory change and bleeding phenotype has been described. Von 
Willebrand factor and FVIII levels increased with age in type 1 patients, with no mitigation in 
bleeding phenotype. In contrast, in type 2 patients von Willebrand parameters do not 
increase with age and ageing in these patients is accompanied by increased bleeding [46].  
1.8. Arteriothrombotic events and bleeding disorders 
Prospective studies have shown that elevations of fibrinogen, FVII and PAI-1 confer a risk of 
atherothrombotic disease, including recurrent myocardial infarction in young men in the case 

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of high PAI-1 [43]. There is little knowledge to whether patients with RBD are protected from 
arterial thrombosis. An Israeli study indicates that severe factor XI deficiency is probably 
protective against ischemic stroke, but not against acute myocardial infarction [47]. In von 
Willebrand disease it has been demonstrated that patients have a lower prevalence of 
coronary heart disease, acute myocardial infarction and ischemic stroke compared to the 
general population [48], whereas there is conflicting evidence as to the risk of cardiovascular 
disease in haemophilia patients [49, 50].   
 
 

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2. RATIONALE  
Because of their extreme rarity very few clinical centers have the opportunity to follow up 
significant numbers of patients; consequently the amount of information available on 
diagnosis and treatment of rare bleeding disorders is very limited.  RBiN will research three 
novel themes - genotype-phenotype correlations, health-related quality of life and innovative 
diagnostics - in a patient population that has not been described to date (the Dutch patient 
population). 
Mapping Dutch individuals with rare bleeding disorders for the first time, including health-
related quality of life, and age related comorbidities 
At present, there is very little information on rare bleeding disorders in the Netherlands, their 
clinical presentation, bleeding score, bleeding episodes, health-related quality of life, 
laboratory parameters, genetics and treatment. Therefore, patients with rare bleeding 
disorders and their doctors have articulated the need for a Dutch study: the Netherlands 
Haemophilia Society (Nederlandse Vereniging van Hemofilie Patiënten, NVHP) and all 
treatment centers are co-initiators of this study. 
Examining genotype-phenotype correlations 
Genotype-phenotype correlations are important in the prognosis and management of 
patients with rare bleeding disorders, especially disorders in which there is a weak or non-
existent association between coagulation factor activity level (laboratory phenotype) and 
clinical bleeding severity (clinical phenotype), i.e. FV, FVII and FXI deficiencies. 
Comparing rare bleeding disorders to haemophilia A and B,  
Often, the rare bleeding disorders are categorized as less severe compared to haemophilia A 
and B. By using similar questionnaires as the upcoming (2017) HiN study (Haemophilia in 
the Netherlands), a direct comparison of the clinical picture of the distinct rare bleeding 
disorders haemophilia A and B is possible.  
Innovations in diagnostics: non-invasive testing, global assays and whole exome sequencing 
Treatment and prophylaxis decisions require frequent venepuncture. Non-invasive testing 
does not exist yet, but would be extremely beneficial to patients and give way to a future 
perspective of home-based monitoring combined with home-based treatment. Saliva 
biomarkers that correlate with anticoagulation status have been identified in coumarin users 
in Radboud university medical center (unpublished data). RBiN will examine whether these 
biomarkers correlate with the clinical and laboratory phenotype in patients with rare bleeding 
disorders and if so, whether we can monitor treatment or rely treatment decisions on these 
biomarkers. 
Precise, personalised treatment of the rare bleeding disorders is hampered by the lack of an 
adequate test that reflects patients’ bleeding potential. At present, treatment decisions are 
based on clinical phenotype, and standard consensus-based approaches which give the 
most accurate indication available, but still need fine tuning. Because the Nijmegen 

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Hemostasis Assay (NHA) measures the key enzymes in both coagulation and fibrinolysis, it 
might give clinicians a more accurate idea of the haemostatic potential of their patients than 
currently used tests can. Ideally, this would enable tailored prophylaxis and ad hoc treatment 
change. 
The diagnosis of a rare bleeding disorder is based upon a step-wise conventional laboratory 
approach as described above (1.4 Laboratory). This strategy is time consuming and 
expensive, due to the high number of laboratory assays that may be needed (sequentially) to 
establish a diagnosis. In addition, repeat testing is mandatory for many assays because they 
are highly variable and dependent on preanalytical and analytical factors. On the other hand, 
the cost of advanced molecular diagnosis such as whole exome sequencing (WES) is falling, 
and its practice is ever more common and developed. In addition, WES can trace more 
genetic abnormalities than current genetic analysis using gene panels can, thereby possibly 
explaining the differences in bleeding tendency in patients with rare bleeding disorders. 
 
 

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3. OBJECTIVES 
Our primary objectives are to: 
 
Describe the epidemiology, clinical presentation, bleeding score, bleeding episodes, 
quality of life, laboratory parameters, genetics and treatment of homozygous and 
known heterozygous individuals (of all ages) with rare bleeding disorders (disorders 
of fibrinogen, FII, FV, FV & VIII, FVII, FX, FXI, FXIII, alpha-2-antiplasmin and PAI-1 
deficiency) in the Netherlands; 
 
Examine the relationship between the clinical and laboratory presentation (clinical 
and laboratory phenotype), and between phenotypes and genetics (genotype); 
 
Examine the relationship between quality of life, phenotype and genotype; 
 
Validate the established factor activity levels for patients to remain without symptoms. 
 
Our secondary objectives are to: 
 
Validate the established factor activity levels for patients to remain without symptoms. 
 
Compare the clinical presentation, bleeding score, quality of life and laboratory 
parameters of individuals with a rare bleeding disorder (disorders of fibrinogen, FII, 
FV, FV & VIII, FVII, FX, FXI, FXIII, alpha-2-antiplasmin and PAI-1 deficiency) to those 
of individuals with haemophilia A or B in cooperation with the HIN-6 investigators 
 
Establish a firm base for a future Dutch registry for homozygous and known 
heterozygous individuals with rare bleeding disorders  
o To develop a standard set of patient-reported, clinical and administrative data 
to be collected on a regular basis 
 
Liaise with the pro-RBDD study, a similar study in Italy, to work towards a pan-
European study linking phenotype to genotype in individuals with rare bleeding 
disorders 
 
To assess if the NHA can distinguish mild clinical phenotypes in patients with similar 
factor activity levels 
 
To evaluate the usefulness of saliva coagulation biomarker tests in the management 
of patients with a rare bleeding disorder 
 
To examine whether age-dependent laboratory changes in factor concentrations and 
fibrinolysis occur in individuals with rare bleeding disorders and if so, whether they 
influence clinical phenotype 
 
To evaluate if patients with rare bleeding disorders are protected from arterial 
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4. STUDY DESIGN 
RBiN is a cross-sectional observational study. All participants will be asked to fill out 
questionnaires, will be interviewed, and asked to provide blood and saliva samples. The 
blood samples will be used for analyses and to set up a biobank.  
 

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5. STUDY POPULATION 
5.1. Population (base) 
Based on the prevalence in the general population of 1:500,000 to 1 per 2 million persons [1, 
2], and a total population of 17 million, there should be 129 to 145 individuals with a 
homozygous rare bleeding disorder in the Netherlands. This could be higher taking into 
account the possible influences of high-risk populations such as Ashkenazi or Iraqi Jews with 
high FXI deficiency rates. To date, 337 patients with a rare bleeding disorder are known to 
the eight Dutch centers that treat such patients. Approximately one third of these patients are 
homozygous; and around two thirds are heterozygous individuals. 1 
All known homozygous and heterozygous patients with a rare bleeding disorder will be 
invited to participate. 
Heterozygous prevalence in the Netherlands have not been evaluated, but can be calculated 
according to the Hardy-Weinberg principle2 to be 93,000 to 99,000 persons, or 0.55% to 
0.58% of the Dutch population (5.5 to 5.8 per 1,000 persons) for all rare bleeding disorders 
combined. Tough the estimated number of heterozygous persons in the Netherlands is much 
higher  than the number of persons known to one of the eight treating centers (~179 
heterozygous patients3), we expect that the individuals with a known heterozygosity 
represent a subset with a more severe clinical presentation, who are therefore of interest to 
our research question.  
Characteristics of the Dutch source population 
The characteristics of the Dutch population (both homozygous and heterozygous) are largely unknown 
and are a part of this study. Numbers of known patients per rare bleeding disorder are presented in  
Table 3 
Table 3. 
 
 
 
                                               
1 A quick inventory in the Radboud university medical center learned that two thirds of patients were 
heterozygous individuals whereas one third were homozygous individuals. Based on an inventory of 
63 medical files performed on July 8, 2016: 65% missing information, 24% heterozygosity, 11% 
homozygosity 
 
2 The Hardy-Weinberg proportion stipulates that for a single locus with two alleles denoted ‘A’ and ‘a’ 
with population frequencies p and q respectively, the expected genotype frequencies are p2 for the AA 
homozygous individuals, q2 for the aa homozygous individuals, and 2pq for the heterozygous 
individuals. The sum of these frequencies must be 1, thus p2 + 2pq + q2 = 1 and p + q=1.  
 

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Table 3 Prevalence of rare bleeding disorders in the Netherlands 
Deficiency  
Estimated prevalence 
homozygosity in general 
population as described in the 
literature [1, 2] 
Estimated prevalence 
homozygosity in the Dutch 
general population 
Patients known at present 
in the Netherlands 
(homozygous/heterozygou
s/unknown) 
Fibrinogen 
1:1 million 
17 
28 
II 
1:2 million 
9 
9 
V 
1:1 million 
17 
22 
V & VIII 
1:1-2 million 
9-17 
4 
VII 
1:500,000 
34 
92 
X 
1:1 million 
17 
10 
XI 
1:1 million 
17 
88 
XIII 
1:1-2 million 
9-17 
16 
PAI-1 
Unknown 
Unknown 
39 
Alpha-2-
antiplasmine 
Unknown  
Unknown 
29 
 
Total 
129-145 
337 
Abbreviations: CI: confidence interval 
$ Based on a population figure of 17,024,133 (www.cbs.nl/nl-nl/visualisaties/bevolkingsteller)  
* Based on a quick inventory of participating sites, which are all sites in the Netherlands licensed to treat rare bleeding 
disorders. Homozygosity or heterozygosity largely unknown 
 
Willingness of patients to participate 
We expect that a high proportion of patients with rare bleeding disorders are willing to 
participate in RBiN. Patients are aware of the rarity of their disorder and the lack of research. 
The Netherlands Hemophilia Society (Nederlandse Vereniging van Hemofilie Patiënten, 
NVHP) is a co-initiator of RBiN and a patient representative takes seat in the steering 
committee. Based on the response rate of the HiN studies we expect a (similar) response 
rate of 70% [51].   
5.2. Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
 
Established homozygous or known heterozygous rare bleeding disorder due to 
deficiency or dysfunction of fibrin, FII, FV, FV & FVIII, FVII, FX, FXI,  FXIII, alpha-2-
antiplasmin and PAI-1  ; 
 
Age 1 year and older; 

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 
For patients ≥ 16 years old; written informed consent. 
 
For patients 12-16 years old; written informed consent from both the patient and their 
parents/legal guardian(s). 
 
For patients <12 years old; written informed consent from their parents/legal 
guardian(s).  
 
5.3. Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation 
in this study: 
 
No informed consent given; 
 
Residency outside of the Netherlands 
 
 

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6. METHODS 
6.1. Study procedures 
Patients will be approached by their own treating physician. Data will be collected through 
questionnaires, a clinical interview, a blood and saliva sample obtained from each 
participant, and thorough review of electronic patient records. An overview of study 
procedures is given in Table 4. All procedures are study related. In case a physician visit with 
the treating physician is already planned, or in case a venepuncture for regular diagnostics or 
treatment is already planned, this can be combined with the study procedures to prevent an 
extra visit.  
Table 4 Overview of study procedures for each adult subject  
Procedure 
Outcome 
Study physician visit 
 
 
Clinical interview 
 
Bleeding score (ISTH-BAT) 
 
RBD bleeding score[52] 
 
Children: iCHEC 
Questionnaires 
 
 
Self-administered 
 
Demographic characteristics (generic) 
 
Socio-economic characteristics (generic) 
 
Clinical characteristics (bleeds, treatment), HIV status (patients born before 1985) and 
hepatitis C status (patients born before 1992) 
 
Medical history (generic) 
 
Sexuality (generic) 
 
General health status (Rand-36/Cho-KLAT) 
 
Sports and physical activity (MAQ/HeptestQ) 
 
Adherence (VERITAS-pro) 
 
Pain (generic) 
 
Needle phobia (generic) 
 
Experience with care (generic) 
 
 
Electronic patient records  
 
 
 
Date of birth 
 
Sex 
 
Baseline factor activity level 
 
Baseline factor antigen level 
 
Treatment type (prophylactic treatment or on-demand treatment) 
 
Body weight and height 
 
Bleeding score at diagnosis 
 
Relevant previous medical history, i.e. arterial thrombosis, atherosclerosis, 
medication use, pregnancies and outcomes 
Blood samples: 72-83 ml 
 
 
Haemostasis 
 
Screening tests 
- 
PFA 
- 
Haemoglobin 

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- 
Thrombocytes 
- 
VWF ristocetin cofactor 
- 
VWF antigen 
- 
VWF collagen binding 
assay 
- 
FVIII act one stage 
- 
FVIII chromogen 
- 
aPTT 
- 
PT 
- 
Fibrinogen 
- 
Thrombin time 
- 
Leucocytes & 
differentiation 
- 
Peripheral blood smear 
- 
Creatinine 
- 
ALAT 
- 
TSH 
- 
D-dimer 
- 
Antitrombin 
- 
Protein C 
- 
Protein S 
- 
TFPI 
 
 
 
Diagnostic tests 
- 
FII 
- 
FV 
- 
FVII 
- 
FIX 
- 
FX 
- 
FXI 
- 
FXII 
- 
FXIII 
 
 
Global assay 
- 
Nijmegen Haemostasis Assay 
 
Fibrinolysis 
 
 
PAI-1 
 
TPA 
 
Plasminogen 
 
α2 –antiplasmin 
 
TAFI 
 
Other 
 
DNA (whole exome sequencing) 
 
Inhibitor assays (only in people ever treated with clotting factors) 
 
Lupus anticoagulans 
 
Anti β2 glycoprotein I 
 
Biobank 
 
2 x 10 ml citrate 
Saliva sample 
 
 
Coagulation 
 
Biomarkers of coagulation 
 
Abbreviations: ISTH-BAT: International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee 
Bleeding Assessment Tool 
6.2. Data collection 
 
Questionnaires 
Patients will receive a personal log on code to an electronic questionnaire after informed 
consent. They will be reminded after one month to fill out the questionnaire online. Patients 
unwilling or unable to fill out the questionnaire electronically will be provided with a hard copy 
of the questionnaire. The questionnaires can be found in separate documents. Children’s 
versions of the questionnaire will be available for children up to 17 years old. Whenever 
possible, questionnaires specifically validated and designed to be filled out by children will be 
used. In general, children from 12-17 years old will be able to fill out the questionnaire 
independently. For children under 12 years old, parents will fill out most of the questions.  
The complete questionnaire will take approximately 40-120 minutes to complete, depending 
on which questions are relevant to the patient. For example, the haemophilia activity list of 
approximately 50 questions will only be filled out by patients with chronic joint impairment, a 

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minority in this patient group. The VERITAS-PRO questionnaire will only be performed in 
patients who are on prophylaxis and patients born after 1992 will not fill out questions about 
hepatitis C and HIV infections. There are also parts of the questionnaire only for women or 
certain age-groups. Therefore, we expect that close to no patients will have to fill out all the 
questions. The questionnaire can be paused at any time and continued later on. 
The patients will submit the questionnaires digitally. The questionnaires will be checked and 
discussed briefly to verify during the patient visit. The bleeding scores are entered directly 
into the eCRF. 
Blood sampling 
All participants will be asked to provide a blood sample during a visit to the haemophilia 
treatment center. Sampling of 1-5% of an individual’s total blood volume is deemed safe 
according to a systematic review of the literature and of existing guidelines[53]. For this study 
we will set the maximum around 2.5% of the total blood volume, or 2mL/kg based on a total 
circulating blood volume of 80mL/kg in persons weighing 2kg or more.  
In adults and children ≥12 years old, roughly 67 ml blood will be drawn through a study-
related venepuncture. Forty-seven ml of this sample will be used for this study while the 
remaining 20 ml will stored in the Radboud university medical center Biobank for future 
research. Additional information about the Biobank is provided in separate documents. 
Blood samples will be used for the experiments specified in table 4. 
In children under 16 years of age, whole exome sequencing will not be performed. Also, the 
following tests are not performed under 12 years of age: ALAT, TSH, creatinine, lupus 
anticoagulans and d-dimer. Therefore, only 47mL of blood will be needed for these children. 
For children under 8 years of age, in addition there will be no blood drawn for the biobank so 
that they only need to supply 27mL of blood.  
Remaining material can be used for future research. In this case, studies will be performed 
according to the protocol Biobank Hematologie HEMBB (IRB Registration number: CMO 
Arnhem-Nijmegen 2013/064). In case the research would not be within the scope of HEMBB, 
the CMO will be consulted in advance. 
Saliva sampling 
Whole saliva will be collected in the morning when patients did not drink more than 2 glasses 
of alcohol on the evening before and did not eat 1 hour before collection. In case of infections 

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in the oral cave or a severe cold, patients will be excluded. Before saliva collection the mouth 
will be rinsed with water and donors will be allowed to chew on 25 cm2 sheet of parafilm 
(Parafilm® M) for 1 minute. Produced saliva will be collected into a 50 ml tube and 
centrifuged at 10,000g for 10 minutes. Samples will be divided in aliquots of 250 µl and 
stored at -80°C. Stimulated whole saliva collection will be used because of its non-invasive 
approach instead of e.g. glandular saliva donation, and since stimulated whole saliva has 
been found to have a large portion of peptides and proteins derived from the parotid glands. 
Specimens will be proceeded with several techniques including ELISA and proteomics.  
Saliva samples are only collected in patients ≥4 years old. 
Electronic patient records  
The clinical data will be collected from the medical charts by the research team using a case 
report form. Each patient will be assigned a unique study code, as described XXXX. A copy 
of the case report form will be sent to the coordinating center (Radboud university medical 
center) for data entry and a copy will be kept at the original center. 
Whole exome sequencing 
In all patients over 12 years of age, whole exome sequencing will be performed. A filter will 
be used to get the results of 135 bleeding related OMIM-proved genes involved in 
haemostasis. 
6.3. Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. The investigator can decide to withdraw a subject from the study for urgent 
medical reasons. 
6.4. Replacement of individual subjects after withdrawal 
Not applicable as all known patients will be invited to participate.  
6.5. Follow-up of subjects withdrawn from treatment 
Not applicable as this study has no follow-up. 
6.6. Premature termination of the study 
If the recruitment of subjects goes unexpectedly too slow within a reasonably time period the 
study ends prematurely. Too slow a recruitment is defined as less than one fifth of known 
patients, thus, less than 60 patients, within one year of the start of the study.  
 
 

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7. STATISTICAL ANALYSIS 
Statistical analysis will be performed by the research team at Radboud university medical 
center. First we describe the general outline of statistical analyses, then we discuss specific 
statistical procedures for the primary and secondary objectives separately, if appropriate.  
In general, continuous variables will be described using standard summary statistics (e.g. 
mean, medians and corresponding 95% confidence interval (CI),); categorical variables will 
be described using frequencies and percentages with their 95% CIs. Group differences will 
be determined using analysis of variance (ANOVA) or Student’s t-test for continuous 
variables and Fisher’s exact test for categorical variables (Fisher-Freeman-Halton exact test 
for variables with more than two categories). Non-parametric tests will be used for results 
with non-normal distributions. Two-sided p-values smaller than 0.05 will be considered 
statistically significant. Because of the explorative character of this study no correction for 
multiple testing will be applied.  
The following parameters will be categorized when appropriate: 
 
 
Bleeding score (ISTH-BAT): ≥5 abnormal for women, ≥3 abnormal for men 
 
Clinical bleeding severity [19]: 
o Asymptomatic: no documented bleeding episodes 
o Grade I bleeding: bleeding that occurred after trauma or drug ingestion 
(antiplatelet or anticoagulation therapy) 
o Grade II bleeding: spontaneous minor bleeding (bruising, ecchymoses, minor 
wounds, oral cavity bleeding, epistaxis and menorrhagia) 
o Grade III bleeding: spontaneous major bleeding (intramuscular haematomas 
requiring hospitalizations, haemarthrosis, bleeding of the central nervous 
system, the umbilical cord or the gastrointestinal tract) 
 
7.1. Primary objectives 
 
Describe the epidemiology, clinical presentation, bleeding score, bleeding episodes, 
quality of life, laboratory parameters, genetics and treatment of homozygous and 
known heterozygous individuals (of all ages) with rare bleeding disorders (disorders 
of fibrinogen, FII, FV, FV & VIII, FVII, FX, FXI, FXIII and alpha-2-antiplasmin and PAI-
1 deficiency) in the Netherlands;  
We will use descriptive statistics using means, SD and medians. Interquartile ranges 
will be used when appropriate. Prevalence will be reported with 95% confidence 
intervals.  
 
Examine the relationship between clinical and laboratory presentation (clinical and 
laboratory phenotype), and between phenotypes and genetics (genotype); 
Descriptive statistics will be used. Regression analysis will be used to determine the 
association between coagulation factor activity level (dependent variable) and clinical 

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bleeding severity as a continuous variable (independent variable), with adjustment for 
age at data collection, sex, etcetera.  
 
Examine the relationship between quality of life, phenotype and genotype; 
Descriptive statistics will be used. Regression analysis will be used to determine the 
association between quality of life (dependent variable) and clinical bleeding severity 
or coagulation factor activity level as a continuous variable (independent variable), 
with adjustment for age at data collection, sex, etcetera.  
 
Validate the established factor activity levels for patients to remain without symptoms. 
Descriptive statistics will be used. 
 
7.2. Secondary objectives 
 
Compare the clinical presentation, bleeding score, quality of life and laboratory 
parameters of individuals with a rare bleeding disorder (disorders of fibrinogen, FII, 
FV, FV & VIII, FVII, FX, FXI,  FXIII and alpha-2-antiplasmin and PAI-1 deficiency) to 
those of individuals with haemophilia A or B (HiN study data will be shared) 
Descriptive statistics will be used. Group differences will be determined using the 
statistics as described in Statistical Analysis above.  
 
Establish a firm base for a future Dutch registry for homozygous and known 
heterozygous individuals with rare bleeding disorders (disorders of fibrinogen, FII, FV, 
FV & FVIII, FVII, FX, FXI, d FXIII and alpha-2-antiplasmin and PAI-1 deficiency): 
o To develop a standard set of patient-reported, clinical and administrative data 
to be collected on a regular basis 
 
      No statistics are involved in this objective. 
 
Liaise with the pro-RBDD study, a similar study in Italy, to work towards a pan-
European study linking phenotype to genotype in individuals with rare bleeding 
disorders 
No statistics are involved in this objective. 
 
To assess if the NHA can distinguish mild clinical phenotypes in patients with similar 
factor activity levels 
We will calculate diagnostic accuracy comparing NHA to ISTH BAT. 
 
To evaluate the usefulness of saliva coagulation biomarker tests in the management 
of patients with a rare bleeding disorder 
Descriptive statistics will be used. Regression analysis will be used to determine the 
association between saliva biomarker tests (dependent variable) and coagulation 
factor activity level as a continuous variable (independent variable), with adjustment 
for age at data collection, sex, etcetera.  

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 
To evaluate the use of WES in explaining differences in bleeding tendency.  
Descriptive statistics will be used.  
 
To examine whether age-dependent laboratory changes in factor concentrations and 
fibrinolysis occur individuals with rare bleeding disorders? And if so, do they influence 
clinical phenotype? 
Descriptive statistics will be used. Regression analysis will be used to determine the 
association between age (dependent variable) and coagulation factor activity level as 
a continuous variable (independent variable), with adjustment for age at data 
collection, sex, etcetera.  
 
To evaluate if patients with rare bleeding disorders are protected from arterial 
thrombosis 
Descriptive statistics for continuous variables will be presented as medians and 25–
75% interquartile ranges (IQR), because data are not normally distributed. For 
comparison of proportions, the chi-squared test or the Fisher’s exact test will be used. 
Wilcoxon tests will be performed for comparison of age and coagulation factor 
levels between groups. Standardized morbidity ratios (SMR) will be calculated to 
estimate the rate of overall arterial thrombosis and of coronary heart disease, acute 
myocardial infarction and ischemic stroke separately in patients relative to that of the 
reference population adjusted for age and sex. 
 

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8. ETHICAL CONSIDERATIONS 
8.1. Regulation statement 
This study will be conducted in accordance to the principles of the Declaration of Helsinki, 
ICH guidelines on Good Clinical Practice (GCP) and in accordance with the Medical 
Research Involving Human Subjects Act (WMO). The local investigator is responsible for 
ensuring that the study will be conducted in accordance with the protocol, ethical principles, 
ICH-GCP guidelines and the WMO. 
8.2. Recruitment and consent 
Subjects will be informed about the study by their treating physician. A minimum of one week 
will be allowed to consider their decision to participate after giving informed consent. The 
patient information letter and informed consent (in Dutch) are attached as separate 
documents. 
8.3. Objection by minors or incapacitated subjects 
For minors the “Code of conduct relating to expressions of objection by minors participating 
in medical research” (“Gedragscode verzet”) is applicable. This code of conduct was 
approved by the Board of the Netherlands Association for Paediatric Medicine (NVK) on 21 
May 2001 and published in NVK Newsletter no. 3, June 2001. 
8.4. Benefits and risks assessment, group relatedness 
There is a possible minor bleeding risk for people participating in this study connected to 
collection of blood. However, this is a very minor risk considering the experience of patients 
and nurses. Blood of patients having RBDs is routinely tested during regular visits to 
treatment centers and risks associated to blood sampling are considered to be negligible.  
Patients will not directly benefit from participating in this study but in the long term this study 
contributes to our knowledge of the rare bleeding disorders and will increase awareness of 
these disorders amongst health care workers, as such improving diagnostics and care. 
Since the population of patients having RBDs is very small, it is important to include all 
patients, also minors (children <18 years), in the study (around one third of known patients 
are minors). Therefore this study may be regarded as group-related. 
8.5. Compensation for injury 
The sponsor/investigator has liability insurance in accordance with article 7 of the WMO. 
The risk associated with the study investigations is considered negligible; therefore no 
additional insurance to compensate for injury (in Dutch: proefpersonenverzekering) is 
necessary and dispensation is requested. 

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8.6. Incentives  
Subjects will not receive any financial compensation or treatment through participation in this 
study. After participation, patients will receive a small present with the study logo, e.g. a 
keychain. Participants will also receive a report of the most important study results. 

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9. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
9.1. Handling and storage of data and documents 
The handling of personal data complies with the Dutch Personal Data Protection Act (in 
Dutch: De Wet Bescherming Persoonsgegevens, Wbp). Patient data will be coded by patient 
study number to protect the privacy of the subjects. Subject identification codes will consist of 
a research center code and serial number. Subject identification codes will be ascribed on 
inclusion and these codes will be used throughout the study. The code will not contain any 
personal identifiers such as date of birth of initials. The key to the identification code will be 
safeguarded by the participating centers for their own patients.  
Data will be stored on protected files on servers according to Standard Operating Procedures 
of the Department of Haematology. Research data will be kept until publication of the study 
results and stored for 15 years. Individual patients will not be identifiable within publications 
or presentations. 
9.2. Monitoring and Quality Assurance  
According to the NFU guidelines this is a study with negligible risk [54]. Therefore there will 
be low-intensity monitoring. 
9.3. Amendments  
Amendments are changes made to the research after a favourable opinion by the accredited 
METC has been given. All amendments will be notified to the METC that gave a favourable 
opinion.  
Non-substantial amendments (e.g. typing errors and administrative changes like changes in 
names, telephone numbers and other contact details of involved persons mentioned in the 
submitted study documentation) will not be notified to the accredited METC, but will be 
recorded and filed by the sponsor.  
9.4. Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the accredited 
METC once a year. Information will be provided on the date of inclusion of the first subject, 
numbers of subjects included and numbers of subjects that have completed the trial, serious 
adverse events/ serious adverse reactions, other problems, and amendments.  
9.5. Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited METC of the end of the study within a 
period of 8 weeks. The end of the study is defined as the last patient’s last visit.  
The sponsor will notify the METC immediately of a temporary halt of the study, including the 
reason of such an action.  

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 In case the study is ended prematurely, the sponsor will notify the accredited METC within 
15 days, including the reasons for the premature termination. 
 
Within one year after the end of the study, the investigator/sponsor will submit a final study 
report with the results of the study, including any publications/abstracts of the study, to the 
accredited METC.  
9.6. Public disclosure and publication policy 
The study results will be disclosed unreservedly, and we aim to report the results in a peer-
reviewed scientific journal. After approval of the study protocol by the METC this study will be 
registered in a public trial registry.  
 

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Appendix 1: Patient information letter and informed consent form