Von Willebrand Disease
Diagnosis of type 1 VWD easy or difficult?
Xiu Yan Jiang, MD
Calgary Laboratory Services
Objectives
1. Understand the current classification of von
Willebrand disease variants and why diagnostic
criteria for type 1 von Willebrand disease remain
controversial.
2. Appreciate the influence of preanalytical variables
upon the laboratory assessment of von Willebrand
factor (VWF).
3. Understand the limitations of currently available
assays of von Willebrand activity.
4. More skillfully interpret functional ratios such as the
ratio of VWF activity to VWF antigen.
Index case
• 8 year-old male with severe epistaxis
requiring cauterization and prolonged bleed
from incision site.
Questions
• What historical information in this case is helpful in
determining whether the patient has a hemostatic
disorder?
• Platelet-type vs coagulation-type bleeding ?
• Based on the information described above, what would
you recommend for initial tests?
• What additional laboratory studies should be done?
• What is the pathogenic mechanism for the likely
diagnosis?
Laboratory Tests
- PT: 11.8 sec
- INR 1.0
- PTT: 35.0 sec
- Fibrinogen: 3.3 g/L
- Ristocetin cofactor activity: 0.49 U/ml (0.41-1.44)
- VWF antigen:
0.77 U/ml (0.40-1.85)
- Factory VIII activity
1.09 U/ml (0.54-1.47)
- CBC: normal
- platelet: 337.4 10x9/L with normal morphology
- Closure time: collagen/EPI >300 sec (84-176)
collagen/ADP 262 sec (63-111)
Discussion
• Review von Willebrand Factor physiology
• Review the classification of von
Willebrand disease
• Discuss the limitations of patient
evaluation
– Medical history and screening tests
– Preanalytical variables
– Review initial laboratory studies used in
evaluation of VWD
Von Willebrand Factor (VWF)
• A large, multimeric blood glycoprotein
• Gene located on chromosome 12
• Synthesized by
– vascular endothelial cells and stores in WeibelPalade bodies
– A small amount synthesized in megarkaryocytes
and stored in the platelet α granules
VON WILLEBRAND FACTOR
Substructure of Human VWF
Hemostatic Functions of VWF
Two major roles in hemostasis
• Supports platelet adhesion and activation at
sites of vascular injury:
– VWF binds extravascular collagen
• Platelets adhere to bound VWF
• Adherent platelets become activated
• Supports coagulation mechanism:
– VWF protects Factor VIII in circulation
– VWF co-localizes Factor VIII at sites of vascular
injury
The Factor VIII/VWF Complex in Plasma
Interaction between VWF, Platelets, and collagen
of the subendothelium
VWD: Definition
NHLBI Expert Panel
• “Von Willebrand disease is an inherited
bleeding disorder that is caused by
deficiency or dysfunction of von
Willebrand factor (VWF), a plasma protein
that mediates the initial adhesion of
platelets at sites of vascular injury and
also binds and stabilizes blood clotting
factor VIII (FVIII) in the circulation.”
The Diagnosis, Evaluation, and Management of
von
Willebrand Disease. NIH publication 08-5832.
2007
The Diagnosis, Evaluation, and Management of
von Willebrand Disease
NIH publication 08-5832. 2007
Classification of von Willebrand Disease
ISTH 2006
• Inherited Quantitative Defects:
– Type 1: Partial quantitative deficiency of VWF
– Type 3: Virtual absence of VWF
• Inherited Qualitative Defects:
– Decreased VWF-dependent platelet adhesion:
• Deficiency of High MW multimers: Type 2A
• Defect not attributable to multimer abnormality: Type 2M
– Increased affinity between VWF and Platelet:
• Abnormal A1 loop of VWF: Type 2B
• Abnormal platelet GP Ib: Platelet-type
– Decreased Factor VIII Binding: Type 2N
• Acquired VWD
Type 1 VWD
Mild quantitative deficiency of VWF
Proposed ISTH Criteria
1. Significant bleeding history
2. Familial history of bleeding
and
3. The bleeding is attributed to low levels of
VWF
~80% of VWD cases are type 1:
Bleeding attributed to “partial VWF deficiency”
Why is the Diagnosis of Type 1VWD
Difficult?
• History: limitations of bleeding history
– Mild bleeding symptoms are common
– In surveys, healthy controls indicate that they have
bleeding symptoms at a similar frequency as
patients carrying a diagnosis of type 1 VWD
• Incomplete penetrance of type 1 VWD
Laboratory evaluation
– Both inherited and acquired factors influence
VWF levels
– There is no single assay to definitely diagnose
VWD
– The correlation between laboratory and
clinical phenotype varies with VWD type
Laboratory Screening
• Ideal screening test
– Sensitive to presence of VWD
– Low false positivity rate
• NHLBI expert panel comment
– There is no ideal screening test currently available
– Panel suggests:
• Initial history and physical
• Laboratory screening with
– Complete Blood Count and platelets
– PT, PTT and fibrinogen
– If bleeding history is strong: consider initial VWF assays
Screening: Utility of PFA-100
Insufficient to either confirm or discard a diagnosis of VWD
• Evaluation of patients with previously diagnosed
VWD
– Sensitivity of PFA: 95%, Sensitivity of BT: 65%
• Performance in clinical practice
– Normal closure time virtually excludes presence of
severe
VWD
– Screening of with bleeding symptoms
• Sensitivity: 62- 80%
• Specificity: 84-89%
• NHLBI Panel comment
Initial Patient Evaluation in VWD
VWD Guideline – NHLBI Expert
Panel
• Quantitative assays:
– VWF ristocetin cofactor (VWF:RCo) activity:
• Functional ability of VWF to bind and agglutinate platelets
in
presence of ristocetin
– VWF antigen (VWF:Ag):
• Immunoassay of VWF protein present
– FVIII activity:
• Coagulant activity of FVIII
• Ratio of VWF:RCo/VWF:Ag
Factors that Affect VWF Level
• Physiologic stimuli elevate VWF level
– Beta adrenergic stimulation
• Stress, trauma, exercise, crying in a frightened child
– Inflammation
– Phase of menstrual cycle
• Lowest during first 4 days of menses
• Intra-individual variation is up to 40%
• importance of timing of the testing with respect to the menstrual cycle is
not clear
– Pregnancy: 3~5 x increased by 3rd trimester
– Thyroid status (Hyperthyroid have higher levels)
• Blood Group (ABO, Lewis):
– The rate of VWF synthesis probably is not affected by blood group;
however, the survival of VWF appears to be reduced in individuals who
have type O blood.
– VWF levels are approximately 25-35% lower in people with
blood group O (Gill JC, et al: Blood 1987; 69: 1689)
Factors that Affect VWF Level
Race: Africans and African Americans have
higher average levels of VWF than the
Caucasian population.
Age: levels increase with age
Importance of repeated testing
Preanalytical Variables
• Sample collection and handling:
– Phlebotomy conditions, Patient stress
level, sample processing, Sample
storage
• Refrigeration or storage of citrated
blood for > 4 hours leads to artifactually
low VWF levels (and loss of HMW
multimers)
• Multimeric analysis:may show abnormal
Limitations of “screening Panel”
• VWF Antigen
– Lower limit of detection usually 1-2 IU/dL
– CV data: Inter-lab usually ≥10%-20%, Intra-lab
~11%
• VWF “activity”
– Multiple assay methods
– Lower limit of detection: Wide range ≤3-20 IU/dL
– CV data*: Inter-lab ≥20%-40%, Intra-lab: ~20%
(especially VWF:Rco), low in immunologic assay
Initial Patient Evaluation in VWD
VWD Guideline – NHLBI Expert Panel
• Quantitative assays:
– VWF ristocetin cofactor (VWF:RCo)
activity:
• Functional ability of VWF to bind and
agglutinate
platelets in presence of ristocetin
– VWF antigen (VWF:Ag)
– FVIII activity:
• Coagulant activity of FVIII
• Ratio of VWF activity/VWF:Ag
Criteria for an abnormal
Activity/Antigen ratio
- 0.7 used in European MCMDM VWD1 study
- 7% of 1166 controls had ratio < 0.7
- 0.6 used in Canadian VWD1 study
- 0.5 is lower limit of range suggested by
NHLBI panel
- Individual labs may need to set their own
criterion
Prototype Laboratory data
Condition
VWF:Ac
VWF:AG
Low or 1
<50
<50
or NL
NL
2A or 2M
<30
<30-200
or NL
<0.5-0.7
2B
<30
<30-200
or NL
< 0.5-0.7
2N
30-200
30-200
<3
<3
3
FVIII
AC/AG
NL
<10
--
Type 1 VWD versus “Low VWF”
JE Sadler, Annu Rev Med 2005; 56:173-191
• NHLBI consensus panel suggested reserving the
diagnosis of VWD type 1 for patients with moderately low
VWF levels
(< 30 IU/dL)
– Facilitates treatment and genetic counseling in this subgroup.
• Suggested “low VWF” when VWF level 30 – 50
IU/dl
– An epidemiological risk factor for bleeding
• Statistics alone predict a high co-occurrence of bleeding
symptoms,
a positive family history of bleeding and low VWF level.
• Using the descriptive term “Low VWF” avoids misdiagnosis of
these patients as having an inherited bleeding disorder
– Still allows appropriate “risk management”
“Functional” VWF by Automated LIA
Tange JI, et al: J Throm Haemost 2009; 7 (supp 2), 533
Tange JL et al: Mayo Clinic “Coagulation Testing Quality Conference 2009
• Automated latex particle-enhanced
immunoturbidometric assay:
– Particles coated with antibody to
“functional epitope”
– Modified procedure: Expand analytical
range to 3 – 1400 IU/dL
– Comparison of VWF-LIA to VWF:Rco
– Decreased CV(%)
VWF Collagen Binding Assay
Another Quantitative VWF Functional Assay
• Quantitates VWF binding to collagen coated
ELISA plates
– More sensitive than VWF:RCo assay to loss of highest multimers
– Lower limit of detection is similar to VWF:Ag
– Has lower CV than VWF:RCo
• Primary site for fibrillar collagen binding is A3
domain
– Highly dependent on the source of collagen, as well as on
whether type 1
collagen or a mixture of type 1/3 collagen is used.
– Four families reported with VWD attributed to defect in A3 domain
VWF Collagen Binding Assay
Another Quantitative VWF Functional Assay
• Role of VWF:CB
– NHLBI panel: “The place of VWF:CB in the diagnosis of VWD
has not been established”
– Patients who have defects in collagen binding may have a normal
VWF:RCo and thus escape clinical diagnosis unless a VWF:CB
assay
is performed
• Availability
• Most routine labs do not offer this assay
• ~ 25% (CV 18-29%)
Genetics of Type 1 VWD
• Type 1 VWD with VWF < 30 IU/dL
– Commonly found inheritable VWF gene defect (75%)
– Most of the mutations: single amino acid substitutions
in domain D3
• Milder Type 1 (“Low VWF”) with VWF 30 – 50
IU/dL:
– Variable inheritance
• VWF gene defect found in 50% of cases
• Blood Group O is more common
• Other genetic factors may play a role
Index Case
• 8 year-old male with severe epistaxis requiring
cauterization and prolonged bleed from incision site
– Closure time: collagen/Epi>300 sec (84-176)
collagen/ADP 262 sec (63-111)
– Ristocetin cofactor activity: 0.49 U/ml (0.41-1.44)
– VWF antigen:
0.77 U/ml (0.40-1.85)
– Factory VIII activity
1.09 U/ml (0.54-1.47)
– VWF activity/Ag ratio: 0.64:1
– Platelet: 337.4 10E9/L
Further Studies
• Platelet aggregation study: slight disaggregation in
1.5 g/l ristocetin
• Multimer study:
– Initial test: relative decreased in high and intermediate
molecular weight VWF
– Repeated test: normal pattern
• Platelet surface markers: CD36, CD41a, CD42a,
CD29, CD31, CD62P, CD42b: normal
• Desmopressin challenge test: good response but the
VWF activity/antigen is still low
(1.21:1.95=0.62:1)
VWD genotype
• Heterozygous A to G transition at
nucleotide 4751 in exon 28 (TYR1584CYS)
of the VWF gene.
• This genetic changes is associated with type
1 VWD.This is the most frequent variant
associated with type 1 VWD.
Family Study
• 13 year-old female (sister) with epistaxis
–
–
–
–
VWF activity (GP1b): 0.60 U/ml (0.41-1.44)
VWF antigen:
0.97 U/ml (0.40-1.85)
Factory VIII activity 1.00 U/ml (0.54-1.47)
VWF activity/Ag ratio: 0.61:1
• Heterozygous A to G transition at
nucleotide 4751 in exon 28 (TYR1584CYS)
of the VWF gene.
Family Study (Mother)
• 41 year-old with menorrhagia
–
–
–
–
VWF activity (GP1b): 0.59 U/ml (0.41-1.44)
VWF antigen:
0.83 U/ml (0.40-1.85)
Factory VIII activity 0.94 U/ml (0.54-1.47)
VWF activity/Ag ratio: 0.71:1
• VWF multimeric analysis: normal range
• Closure time Collagen/EPI >250 seconds (84176)
• Heterozygous A to G transition at nucleotide 4751
in exon 28 (TYR1584CYS) of the VWF gene.
Specific Assays Used to Diagnose
and Classify VWD:
• Initial quantitative screening (NHLBI guideline)
– VWF:RCo: Function of VWF to bind/agglutinate platelets
– VWF antigen: Immunoassay of VWF protein
– FVIII activity: Coagulant activity of FVIII
• Ratio of VWF:RCo/VWF:Ag
• Supplemental studies: Important to define Type 2
VWD
– VWF multimer: Size distribution of VWF polymers
– VWF:CB: Function of VWF to bind collagen
– Other functional assays: LD-RIPA, VWF:FVIII binding
– VWF gene analysis
– Platelet-binding studies
– Assays for antibodies to VWF
Treatment
• Desmopressin
–
–
–
–
Treatment of choice in type 1 vWD
Milder responses in type 2A and 2M
In type 2B vWD, causes worsening of thrombocytopenia
Ineffective in type 2N and 3 vWD
• vWF/Factor VIII Concentrates
– Indicated in type 2N and 3 vWD and in subtypes with
more severe phenotype
• Antifibrinolytic agents
– Tranexamic acid
– Adjunctive therapy or as sole therapy in mild bleeds
Conclusions
Diagnosis of type 1 VWD easy or difficult?
• Screening tests such as the PFA-100 are insufficient
to include or exclude von Willebrand disease
– One should assay VWF:Ag, VWF:activity and F-VIII
• Physiologic factors and sample processing are
important pre-analytic variables that influence
diagnostic value of clinical assays
• The Panel emphasizes the importance of the timing of
the phlebotomy for assays, with the patient at his/her
optimal baseline as far as possible.
• Type 1 VWF is a quantitative defect.
– The distinction between diagnoses of “Low VWF”
and Type 1 VWD is still being debated
• VWF Activity and VWF:Ag ratio may be an important
clue for diagnosis.
• Supplemental test of VWF are useful to evaluate or
exclude type 2 VWD