Von Willebrand Disease (Green Top 2017, ISTH 2021 & BSH 2024)

N.B. Beware! The ISTH 2021 guideline has made small but exam-notable changes to diagnostic testing pathways compared to the previous BSH guideline.

Intro

The most common inherited bleeding disorder. Predominantly attributable to reduced levels of VWF activity, frequently but not always attributable to a defect in the VWF gene.

Common presenting complaints:

Mucocutaneous bleeding
Postpartum haemorrhage
GI / tooth extraction / menorrhagia / minor wounds / surgical bleeding

Von Willebrand Factor

Large, complex glycoprotein (1000-20,000 KDa) essential for normal haemostasis

Half-life: 12 hours

VWF gene located on short arm of Chromosome 12

(Note: There is a pseudogene on Ch. 22 —> complicates interpretation of genetic testing!)

Synthesized in:

Vascular endothelial cells (and stored in Weibel-Palade bodies)
Megakaryocytes and so it is present on platelets

Produced as large multimers that are then degraded by ADAMTS13

Two roles:

Carrier protein for FVIII —> prolongs FVIII half-life by preventing degradation in plasma
Adhesive protein involved in interaction between platelets and vessel walls

Diagnostic Levels

VWF levels are a guide only and not a strict reference for diagnosis

VWF levels often rise with age —> Antigen levels <50 IU/dL normalise in >40% of individuals

Normal Range VWF Antigen

40-240 IU/dL (25% lower in Group O individuals)
Reference ranges should be generated from 120 local healthy donors, or local verification of manufacturer’s ranges

Von Willebrand Disease

Activity 30 IU/dL + mucocutaneous bleeding

Probable VWD

Incidental finding of activity <30 IU/dL

‘Low VWF in a 1o haemostatic deficiency’ (ISTH) / ‘Mild VWF Reduction’ (BSH)

Activity 30-50 IU/dL + bleeding history
Requires investigation for other haemostatic defects

ISTH 1994 Saddler Classification

Does not define or predict response to treatment and has a variable relationship to genetics. There two groups:

Type 1 & 3 - Quantitative reductions in VWF levels
Type 2’s - Qualitative reductions in VWF activity

Type 1 (Autosomal Dominant)

Quantitative, partial deficiency of VWF
Includes mutations that cause rapid clearance of VWF, e.g. Type 1c Vincenza.
Autosomal dominant inheritance when VWF <30 IU/dL (>30 due to variable penetrance).

Type 1c Vincenza

Causes increased rate of VWF clearance —> short, limited response to desmopressin (assessed using a desmopressin trial with levels at 1 and 4 hours post administration).
Ix: Genetics, VWF propeptide levels

Type 2A (Autosomal Dominant)

Reduction in VWF-dependent platelet adhesion and absent HMW multilmers

Type 2B (Autosomal Dominant)

Gain of function defect: Abnormally increased affinity of VWF to Gp1b platelet receptors
Results in a mild consumptive thrombocytopenia, made worse by use of desmopressin

Platelet-Type VWD (?Inheritance)

Abnormally increased responsiveness of Gp1b platelet receptors to VWF
The defect is in the GP1BA gene, not in the von willebrand factor.

Type 2M (Autosomal Dominant)

Reduction in VWF-dependent platelet adhesion, without a reduction in HMW Multimers

Type 2N (‘Normandy’) (Autosomal Recessive)

Variants in the FVIII binding region of VWF —> Reduced binding affinity of VWF to FVIII —> Shortened FVIII half-life —> low FVIII:C relative to VWF:Ag
Separate, but difficult to distinguish, from mild haemophilia A

Type 3 (Autosomal Recessive)

Virtually complete deficiency of VWF.
25-50% of carriers also have a bleeding phenotype

Diagnostic testing - Summary (based on isth 2021)

N.B. Genetic testing is increasingly used, expect this to continue to move higher up the flowchart. Some centres may already be using it at an earlier stage than represented here. The range of available VWF activity assays has expanded, the ‘VWF:Rco’ is no longer first choice in some labs.

Diagnostic testing - Details

A standardised bleeding assessment tool (BAT) - such as this - is useful for screening patients and helps to determine the pre-test likelihood of a patient being diagnosed with a significant bleeding disorder. A low score helps support a decision not to proceed with further laboratory testing (in conjunction with history and examination).

Pre-Analytical variables

VWF levels rise with inflammation, anxiety (eg needle phobia), exercise and pregnancy

Pre-analytic variables account for >70% of laboratory errors

Sample haemolysis, icterus and lipaemia may affect VWF assays, depending on method used

—> Tests should be performed at least twice on separate occasions before making diagnosis

Sample Preparation, Storage & Testing

Whole blood citrated samples should be stored/transported at ambient room temperature for a maximum of 12 hours to avoid pre-analytic drop in VWF and FVIII:C

Samples are centrifuged to separate plasma from cells and should be tested within 4 hours of separation (and still within total of 12 hours since phlebotomy) to avoid pre-analytical drop in FVIII:C (or frozen within 4 hours for testing at a later date)

There can be significant interlaboratory variation, most pronounced the lower the VWF level. Consider referring all testing to regional specialist laboratories

Primary Diagnostic Tests

Full blood count - Platelet count and mean platelet volume (MPV)

PT, APTT & FGN - APTT may be normal or prolonged

Blood Group? - VWF levels 25% lower with Group O but there are no group-specific reference ranges and bleeding phenotype is the same regardless of blood group —> no benefit/need to check blood group.

FVIII:C

APTT-based 1-Stage Assay or Chromogenic Substrate Assay (CSA)
FVIII half-life regulated by VWF and so level may be low in all types
May be normal

Plasma VWF Antigen Level

ELISA - Rabbit anti-VWF binds to VWF in patient plasma and produces colour reaction
Or Latex Particle Agglutination (LIA, e.g. Accustar) – Latex particles with Anti-VWF bind to VWF in patient plasma and cause agglutination.
Or Chemiluminescent imunoassay (CLIA)
Pros and Cons to different assays (details in BSH guideline), e.g. falsely high levels can occurs with LIA and CLIA in presence of Rheumatoid Factor

VWF Ristocetin Cofactor (VWF:Rco) Activity

Ristocetin binds to the VWF A1 domain causing a conformational change in VWF which aids VWF binding to platelet GP1b receptors, resulting in platelet crosslinking.
The test reflects VWF binding to platelet GP1b-V-IX by assessing ristocetin cofactor activity.
It measures the agglutination of platelet in a solution containing an excess of ristocetin along with dilutions of patient plasma. The activity level is determined by comparison to a reference plasma.
Result is also dependent on presence of HMW multimers and intact GP1b binding sites.
Poor accuracy at low levels
May be spuriously affected by benign VWF variants, present in 0.1-0.5% of population
Non-physiological test – some patients will test low in the absence of a bleeding phenotype.

Newer alternative assays for activity of VWF binding to platelet GP1b

Includes VWF:GP1bM, VWF:GP1bR, VWF:Ab
These assays use combination of ristocetin with either wild-type or mutated recombinant GP1b-fragments
May be spuriously affected by benign VWF variants, present in 0.1-0.5% of population
Still non-physiological —> same issue as VWF: Rco that some pts will test low in absence of bleeding Sx

Secondary Tests when Activity:Antigen ratio is <0.7

VWF Collagen binding (VWF:CB) Assay

For use in suspected cases of type 2A/2B/2M
Or as a potential additional test for individuals with normal first line tests and no other causes for bleeding disorder identified
Principle: Collagen binds to the VWF A3 domain —> Successful binding is dependent on the presence of intact HMW multimers —> VWF:CB is sensitive to a loss of HMW multimers
The VWF:CB to VWF:Ag ratio may be used as an (inferior) alternative to multimer analysis
Tested by chemiluminescent immunoassay (HemosiL AcuStar at Addenbrookes).
- Uses particles coated with collagen peptides.

Multimer Analysis

For use in suspected cases of type 2A/2B/2M
Gel electrophoresis combined with visualisation + quantification methodologies
Cons: High error rates, up to 15%, when monitored by external quality assessment (EQA)
Cons: There are rare but multiple exceptions to the rules when using multimer analysis for typing VWD (see BSH guideline for details)
Note: Ultra-large MW multimers can be seen on multimer analysis with Type 1C, Type 2M and TTP

VWF binding to FVIII (VWF:FVIIIB)

For use in suspected cases of Type 2N VWD
Variants in the FVIII binding region of VWF —> low FVIII:C relative to VWF:Ag
VWF:FVIIIB/VWF:Ag ratio (VWF:VIIIB ratio)
- <0.3: Likely homozygous / compound heterozygous
- 0.3-0.7: Likely heterozygous
Cons: There is currently no international standard for this test and no UK EQA scheme —> therefore genetic testing should be performed in parallel with this assay

Ristocetin-Induced Platelet Agglutination (RIPA)

In normal individuals, low concentrations of ristocetin are insufficient to initiate VWF-dependent platelet agglutination
If agglutination does occur at low concentrations of ristocetin (0.5mg/ml) it suggests the pathological enhancement of VWF-Gp1b interactions seen in Type 2B VWD or Platelet-Type VWD.
If performed, do so in parallel with genetic testing

Genetic Analysis

Increasingly appropriate and should be considered as an aid in many circumstances
See below

Genetic testing

VWF Gene

VWF gene found on chromosome 12. Contains 52 exons.

(Note: There is a pseudogene on Ch. 22 —> complicates interpretation of genetic testing!)

VWF Gene Mutations

Type 1 & 2 VWD – usually autosomal dominant, with majority due to missense mutations.

Type 3 & 2N – usually autosomal recessive, with wide varieties of different mutations

Huge number of variants have been reported and most are yet to be differentiated into pathogenic vs benign

Mosaicism is uncommon but reported

Unaffected parents of affected offspring, where the parent is not found to carry the familial variant (gonadal mocaicism)
Unaffected parents of affected offspring, where the parent does carry the familial variant but at a reduced % in blood and mucosa (somatic mocaicism)

Genetic Testing in UK in 2024

Index cases of VWD should have genetic testing performed after laboratory assays confirmed on two occasions

Informed consent required prior to any genetic testing

R121 (single gene) or R90 (Bleeding and Platelet Disorders panel) via NHS National Genomic Test Directory

Most UK testing currently performed by NGS or direct DNA sequencing

Large minority of patients will have no identifiable mutation

Diagnosis in Children and Infants

FVIII levels are high after birth

Difficult to obtain non-activated, uncontaminated samples

Stress increase the VWF level

--> Try to wait until 6 months of age before testing

Treatment

Tranexamic Acid

Good for minor surgery and minor bleeding

Good solo or in combo with desmopressin

Topical, mouthwash, oral or IV available

Desmopressin

SE: Flushing, hypotension, fluid retention, hyponatraemia, aterial occlusion

Safe use:

Fluid restrict to 1L in 24 hours after dose
Monitor Na if <2 y.o.
Avoid use if atherosclerosis
Note response to a 2nd dose is 30% lower

Factor Concentrates

Consider when desmopressin is contraindicated or proven to be ineffective

Usually combined with FVIII - this is to remove the lag time associated with using pure VWF concentrate. I.e. It could take up to 12 hours post dose to see a clinical response to pure VWF concentrate due to the time taken for the VWF to circulate and pick up free FVIII.

As a result, be aware of FVIII accumulation with repeated doses due to variable half-lives

VWF:Rco and FVIII levels rise by approx. 2 IU/dL per iu/kg administered

Wilate – 1:1 ratio FVIII:VWF. Plasma derived. Half life 12 hours.

Voncento – 1:2.4 ratio FVIII:VWF. Plasma derived. Half life 12 hours.

Willfact – high purity VWF content. Plasma derived. Not commonly used in UK.

Veyvondi (Vonicog Alfa) - 1st recombinant product. Purely VWF so need to consider co-administration with a recombinant FVIII if FVIII <40%. Half life 18 hours. Raised VWF 2% for every 1u/kg given. Currently available in UK for bleeding and pre-op planning but not for prophylaxis, and only reimbursed when prescribed from a Comprehensive Care Centre (as of 2022).

Surgery & Dentistry

Minor Surgery

Aim VWF:RCo & FVII levels >50 IU/dL

Major Surgery (e.g. Hip replacement)

Raise VWF:RCo & FVII levels to >100 IU/dL pre-op
Maintain VWF:Rco or FVIII >50 IU/dL for 2 weeks, then >30 for further 2 weeks

Response to desmopressin should be measured prior to proceeding with the op

Factor Concentrate Prophylaxis

30-50 iu/Kg 2-3 times per week in children with type 3 who develop joint bleeding

Not often required, except in patients with Type 3 VWD or joints bleeds

76% of type 3, 17% type 2 and 7% type 1 patients receive prophylactic concentrate

Other measures

Mirena coil / OCP

Platelet transfusion if bleeding persists despite the above and VWF activity is corrected

Pregnancy Plan

Inhibitors

VWF Inhibitors in VWD

5-10% of type 3 patients who have been multiply transfused
Caused by IgG antibodies that are not time or temperature dependent
Unlikely to occur in type 1 or 2
Present with loss of response to concentrate, or sometimes with anaphylaxis
Rx: rFVIII, rFVIIa, Platelet transfusion, TXA

Inhibitor Screen

HemosiL AcuStar at Addenbrookes
Incubate patient plasma with a source of VWF, allowing for antibody-antigen interaction
Then test VWF functional activity of the sample
Result is compared to a control plasma incubation
Decreased function in the patient sample relative to control suggests presence of inhibitor

Acquired VWD

Causes

Immune

Hypothyroidism
Myeloma, Waldenstroms

Consumptive

MPN due to thrombocytosis
Mechanical – Aortic stenosis, LV Assist Device, Vavle replacement, ECMO

Wilms tumour

Drugs

Ciprofloxacin

Treatment of avwd

Treat the cause

VWF concentrates

Desmopression

IgM paraprotein

Plasma Exchange / Immunoabsorption

IgG paraprotein

IVIg