VWF:RCo

UPDATE ON VON WILLEBRAND FACTOR TESTING:
COMPARISON OF NEW AND OLD ASSAYS FOR DIAGNOSING
AND MONITORING VON WILLEBRAND DISEASE
Catherine P. M. Hayward, MD PhD, FRCP(C)
Head, Coagulation, Hamilton Regional Laboratory Medicine Program
Professor, Pathology and Molecular Medicine, and Medicine
McMaster University, Hamilton, Ontario, Canada
Financial Disclosures
Catherine P. M. Hayward
• No financial or other conflicts of interest to disclose
Perspectives – von Willebrand Disease
One of the most common bleeding disorders
overall prevalence of ~1/1000, 90% congenital
Much is known about pathogenesis of von Willebrand
factor defects
• Particularly qualitative defects
Laboratory testing
• important for diagnosis and therapy monitoring
Diagnosing mild quantitative defects can be challenging
• “Gold standard” assays with high coefficient of variation
3
Diagnosis of VWD
• History
• Mucocutaneous bleeding
• Family history of bleeding
• Laboratory results
• Factor VIII activity
• VWF antigen (VWF:Ag)
• VWF activity assay
• VWF ristocetin cofactor activity (VWF:RCo)
• VWF collagen binding activity (VWF:CB)
• Newer assays: eg Innovance® VWF activity (VWF:Ac)
• VWF multimer analysis
• Molecular testing
Expected Laboratory Findings in VWD
Figure from NHLBI VWD Guidelines (2008) kindly provided by W. Nichols
Normal
Type 1
Type 2A
Type 2B
Type 2M
Type 2N
Type 3
PLT-VWD*
VWF:Ag
N
L,  or 
 or L
 or L
 or L
N or L
Absent
 or L
VWF:RCo
N
L,  or 
 or 


N or L
Absent

FVIII
N
N or 
N or 
N or 
N or 

1-9 IU/dL
N or L
RIPA
N
Often N

Often N

N
Absent
Often N
Absent
Absent
Absent

Absent
Absent
Absent

PFA-100 CT
N
N or 



N


BT
N
N or 



N


Platelet
count
N
N
N
 or N
N
N
N

N
N
Abnormal
Abnormal
N
N
Absent
Abnormal
LD-RIPA
VWF
multimer
pattern
Prototypical cases; exceptions exist. *PLT-VWD, platelet-type VWD
CP1279395B-1
VWF:RCo
• Commonly performed on an aggregometer
• Reagent – antibiotic not involved in VWF physiologic
function, from a sole manufacturer
• recognized lot-to-lot variability
• Historic “Gold standard”
• Poor precision
• Problem for diagnosing both quantitative and qualitative
defects
• Inadequate lower limit of detection
• Impact: diagnostic errors and misclassifications
Further VWD Diagnostic Challenges
• Ethnic-specific sequence variations in VWF gene
incorrectly reported as mutations
Bellisimo et al, Blood 2012;119:2135-40
• Some variants (e.g., D1472H, which is quite common)
are not associated with bleeding but reduce VWF:RCo
~ 28%
• D1472H does not impair VWF binding to recombinant,
mutant GPIba in research assays
Flood et al, Blood 2010;116:280-6
Flood et al, Blood 2013;121:3742-4
Important Issues in VWD Testing
Are there better tests for diagnosing and monitoring VWD?
Direct comparison studies
many
few have not addressed therapy monitoring, or impact on VWD classification
None have been prospective, superiority/non-inferiority studies
require a large number of subjects
Are there better approaches/tests to distinguish:
type 1 from type 2 VWD?
type 1 from type 3 VWD?
defects associated with accelerated clearance from other forms of VWD?
Are there better approaches for monitoring VWD therapy?
Example of a real problem
Patient with known type 2B VWD and GI bleeding
• Longstanding mucocutaneous bleeding problem
• Unnecessary splenectomy prior to diagnosis of type 2B VWD
• Laboratory findings:
• Thrombocytopenia – estimated platelet count 50 X 109/L
• VWF:Ag 0.85 U/mL
• VWF:RCo 0.62 U/mL
• Multimers: loss of high molecular weight multimers
• RIPA: increased aggregation with 0.5 mg/mL ristocetin
• Type 2B VWD confirmed by genetic testing
• heterozygosity for c.4378C>T [p.(L1460F)]
VWF levels during treatment of this bleeding episode
Resident asks: ...what level are you aiming for? Why is she still bleeding with normal VWF levels?
You indicate that these are great questions and wonder about alternatives
Technical Innovations in VWF Testing
• Increased automation of activity and antigen
assays
• ease of testing
• Assay modifications to improve:
• Precision
• Lower limit of detection
• Use of additional tests:
• VWF propeptide (pp) & VWFpp/VWF:Ag ratio
• Functional assessment of VWF binding to collagen type VI
• Functional assessment of VWF binding to glycoprotein Iba
Technical Improvements: VWF Antigen Assays
Methodology
Imprecision
Limit of Detection Special
(coefficient of
considerations
variation, %)*
ELISA
10 - 20% [6]
0.02 IU/mL [6]
Immunoturbidometric 2.6 – 3.0% [27] 0.05 IU/mL [6]
assays
0.02 IU/mL [11]
0.022 IU/mL [27]
Chemiluminescent
7% [26]
0.005 IU/mL [26]
assays
3.9-5.3% [27] 0.003 IU/mL [27]
Not automated.
Declining use.
Not available in some
countries.
Requires an ACL
AcuStar instrument
(Instrumentation
Laboratory, Bedford
MA)
VWF Activity Assay Modifications
• VWF:RCo Modifications:
• Quantification by enzyme-linked immunosorbent
assay (ELISA), immunoturbidometric or
chemiluminescent endpoints
• Replacement of target platelets with immobilized,
wild-type GPIba, with detection by an
immunoturbidometric or chemiluminescent endpoint
Technical Improvements: VWF Activity Assays
Methodology
Imprecision
Limit of
(coefficient of Detection
variation, %)*
Assays using ristocetin
Agglutination
20-40% [6, 25] ~0.10 – 0.20
IU/mL [6, 11, 24,
29]
Immunoturbidometric 3.8-6.2% [27] 0.03 IU/mL
<3.0% - <3.5% (modified assay)
[40]
[11]
0.04 IU/mL [27]
Chemiluminescent
7% [26]
0.002 IU/mL [26]
4.2-6.9% [27] 0.005 IU/mL [27]
Special
considerations
Limitations associated
with using ristocetin.
Declining use.
Limitations associated
with using ristocetin
Not available in some
countries. Requires an
ACL AcuStar
instrument. Limitations
associated with using
ristocetin
Newer Functional Assays That Assess VWF
Binding to Platelets without Ristocetin
• Quantify VWF binding to a mutant form of its platelet
receptor GPIba (Flood et al, Blood 2011;117:e67-e74).
• Some such assay now available commercially
• Potential advantages over VWF:RCo?
• Not affected by polymorphisms that only affect ristocetin-dependent
VWF function
• Don’t require an antibiotic made by a single manufacturer, with
known lot to lot variability
• Precision
• Lower limit of detection
Technical Improvements: VWF Activity Assays
Methodology
Imprecision
Limit of
Special
(coefficient of Detection
considerations
variation, %)*
Assays using a gain-of-function glycoprotein Iba mutant
ELISA
10-20% [6]
0.02 IU/mL [6]
Not commercially
available
Immunoturbidometric 5.6% [29]
0.05 IU/mL [40]
Not available in some
0.04 IU/mL [29]
countries.
Gives lower results
than VWF:RCo for
some VWD subjects
[29]
Hamilton Study
• Evaluated Siemens Innovance® VWF Ac
(VWF:Ac)
• Potential advantages over VWF:RCo (gold standard)
• Promising data reported by Lawrie and colleagues
• Had not assessed VWD classification or therapy monitoring
• Major goals of our study:
• assess the utility of the assay for VWD diagnosis, and
therapy monitoring, after validating that the assay could
be performed on an instrument from a different
manufacturer
Methods
Subjects
• 100 healthy controls
• 262 consecutive clinical samples from 217 patients
referred for VWF testing
• Subjects: 197 adults, 64 children, n=1 age unknown
• One was a subject with type 2B VWD that we asked to donate a
sample as she had repeated normal VWD screens using
VWF:RCo
• Chart reviews to retrieve clinical information for
Hamilton patients, including results of genetic
investigations, if available
Methods
Measured
• VWF:Ac, VWF:RCo and VWF antigen (VWF:Ag)
• Compared results of these assays
• VWF:Ac
• Results of tests done in Hamilton and Ottawa were compared as
part of validating that the assay could be run on a different
manufacturer’s instrument
• Compared ratios of activity/antigen
• Two hematologists: independently interpreted test
results of subjects with one or more results that were
outside reference intervals (RI)
Exchanged Samples (n=58) Tested on Different Manufacturer’s Instruments
1.40
y = 0.90x + 0.04
R² = 0.96
VWF:Ac (Sysmex CS2000i) (U/mL)
1.20
1.00
0.80
0.60
0.40
0.20
0.00
0.00
0.20
0.40
0.60
0.80
1.00
VWF:Ac (STA-R Evolution) (U/mL)
1.20
1.40
Validated Reference Intervals
assay on STA-R: 0.48-1.80 IU/mL
manufacturer’s RI 0.48-1.73 IU/mL
no significant gender differences in findings
Precision and Lower Limits
CV significantly lower for VWF:Ac than VWF:RCo
• normal sample:
• abnormal sample:
5.6% versus 13.0%, p<0.005
9.4% versus 14.5%, p<0.005
Lower limits of VWF detection were also significantly different
• VWF:Ac
0.04 IU/mL
• VWF:RCo 0.10 IU/mL, p<0.005
Cutoffs (based on healthy controls)
• Ratio VWF:Ac/VWF:Ag
• Ratio VWF:RCo/VWF:Ag
0.63 IU/mL
0.70 IU/mL, p<0.001
Additionally, both activity assays were affected by
preanalytical errors (e.g., chilled sample)
Healthy Control Data: correlations
A
B
3.0
2.8
2.8
y = 0.99x - 0.12
R² = 0.68
2.6
2.4
2.4
2.2
2.2
VWF:Ac (U/mL)
VWF:Ac (U/mL)
2.6
3.0
2.0
1.8
1.6
1.4
1.2
1.0
y = 0.94x - 0.05
R² = 0.73
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0
VWF:RCo (U/mL)
VWF:Ag (U/mL)
Difference in Activity (VWF:RCo – VWF:Act)
Difference
Bland-Altman Plot – Data for all subjects
estimate of mean difference very close to value reported by Lawrie et al
Mean difference 0.07 (95% CI 0.04 to 0.10)
Agreement limits -0.49 to 0.63
1
.8
.6
.4
.2
0
-.2
-.4
-.6
-.8
-1
0
.2
.4
.6
.8
1
1.2
1.4 1.6
Average
1.8
2
2.2
2.4
2.6
Average of VWF:Act and VWF:RCo (U/mL)
2.8
3
Clinical Samples
significant proportion with VWF:Ac<VWF:Ag
A
5
4
4.5
3.5
3
2.5
2
1.5
1
y = 0.62x + 0.14
R² = 0.65
3.5
VWF:Ac (U/mL)
4
VWF:Ac (U/mL)
B
y = 0.79x + 0.06
R² = 0.78
3
2.5
2
1.5
1
0.5
0.5
0
0
0
0.5
1
1.5
2
2.5
3
3.5
VWF:RCo (U/mL)
4
4.5
5
0
0.5
1
1.5
2
2.5
VWF:Ag (U/mL)
3
3.5
4
VWF:Ac by subject age: overlap amongst adults and children
treatment samples excluded
3.25
3.00
pediatric
Series1
2.75
adult
Series2
2.50
VWF:Ac (U/mL)
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
0
10
20
30
40
50
Age (years)
60
70
80
90
100
Subjects with known VWD, not on therapy
A
B
1.0
Series1
type 2A
0.9
Series1
type 1
0.9
0.8
Series2
type 3
0.8
Series2
type 2B
0.7
Series3
type 2N
0.7
Series3
type 2M
0.6
0.5
0.4
VWF:Ac (U/mL)
VWF:Ac (U/mL)
1.0
0.6
0.5
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
VWF:RCo (U/mL)
VWF:RCo (U/mL)
In most patients with types 2A, 2B or 2M VWD, VWF:Ac was
undetectable and/or significantly lower than VWF:RCo
VWD Treatment Samples
5
4.5
Series1
1 (W)
3.5
Series2
2B (H)
3
Series3
2M (H)
VWF:Ac (U/mL)
4
2M (W)
Series4
2.5
3 (H)
Series5
2
acquired
Series6
1.5
1
0.5
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
VWF:RCo (U/mL)
type 2B VWD subject on replacement had much lower activity estimated by VWF:Ac
Abbreviations: W, Wilate; H, Humate P.
Acquired VWD subjects were treated with IV IgG (all had an IgG paraprotein)
3
Interpretations based on VWF:Ac
type 3
type 2 (A,
B or M)
4
type 2A or
2B
type 2M
1
5
1
6
6
3
6
9
4
16
type 1 , 2M
not
excluded
type 1
normal /
low VWF
1
1
normal/
type 1
low VWF
type 1,
type 2A type 2 (A,
2M not type 2M
or 2B
B or M)
excluded
Interpretations based on VWF:RCo
type 3
3
Interpretations based on VWF:Ac
type 3
type 2 (A,
B or M)
4
type 2A or
2B
type 2M
1
5
1
6
6
3
6
9
4
16
type 1 , 2M
not
excluded
type 1
normal /
low VWF
41%
interpreted
differently
1
1
normal/
type 1
low VWF
type 1,
type 2A type 2 (A,
2M not type 2M
or 2B
B or M)
excluded
Interpretations based on VWF:RCo
type 3
Results for subjects with D1472H polymorphism
• Based on bias estimates (of -7%), the average VWF:Ac
values should be lower in these subjects (n=6), if there
were no differences in VWF activity measured by VWF:Ac
and VWF:RCo assays
Findings (n=6 subjects with D1472H):
• VWF:RCo 0.76±0.20 IU/mL, 0.61-1.14
• VWF:Ac
0.79±0.04 IU/mL, 0.75-0-83
• p=0.35
Study Findings
• The Innovance® VWF Ac is acceptable alternative to the
•
•
•
•
•
VWF:RCo for the diagnosis and monitoring of VWD
This new assay has an improved precision and a lower limit of
detection than the VWF:RCo
Some patients with VWD have much lower VWF activity
measured by the Innovance® VWF Ac than VWF:RCo
Using the Innovance® VWF Ac instead of VWF:RCo, increased
the number of cases considered to have qualitative VWF
defects, including those with a loss of HMWM
Large prospective studies would be needed to determine if
using the Innovance® VWF Ac in VWD screens improves the
detection, classification and monitoring of VWD
Would be interesting to further explore relationships between
Innovance® VWF Ac findings and VWF mutations
Back to the patient with type 2B VWD & GI bleeding
VWF:Ac Results
Acknowledgments
• Lukas Graf
• Karen Moffat
• Steve Carlino
• Alfonso Iorio
• Anthony Chan
• Antonio Giulivi
• Bob Montgomery
• D1472H samples
• Siemens Healthcare
• Assay kits
Springtime Bliss
 Cathy Hayward

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