Automated vWF
Assay vs. Ristocetin
Cofactor Method in
Diagnosis of von
Willebrand Disease
Group 3 Presentation
Robert Ackerman
Sean Cavanagh-Voss
Jennifer Le
Gonzalo Ortiz
Shea Taylor
Autumn Walker
Evidence-Based Clinical Reasoning I
USF Morsani College of Medicine
February 2014
Case Description
 A 21 year old woman presents to your office for evaluation of
abnormal menstrual periods. Her periods last from 7 to 9 days
requiring up to 10 pads a day (excessive). On further
questioning, she admits to easy bruising and nosebleeds.
Family history reveals that her mother has had similar
problems.
 You suspect von Willebrand disease (vWD). vWD is the most
common inherited coagulation abnormality.
 Your laboratory informs you of a new automated assay for vW
factor (vWF) activity. Since you have not heard about it, you
decide to check what the published literature says about this
new test and how does it compare to the established ristocetin
cofactor method that you used to order to confirm the diagnosis
of vWD.
von Willebrand Disease (vWD)
 Deficiency in von Willebrand factor (vWF)
– Two main functions of vWF:
• Binds and stabilizes factor VIII
• Mediates platelet adhesion to vascular injury site
– Three classifications of vWD:
• Type 1: partial, quantitative deficiency of vWF
• Type 2: qualitative abnormalities of vWF (defective
platelet adhesion or factor VIII binding)
• Type 3: near absence of vWF
Diagnosis of von Willebrand Disease
 Three main criteria:
– Past medical history of excessive bleeding
– Family history of excessive bleeding
– Laboratory evaluation of vWF
• Screening tests
– Platelet count
– Activated partial thromboplastin time
– Bleeding or closure times
• Confirmatory tests
– von Willebrand factor antigen (VWF:Ag)
– von Willebrand ristocetin co-factor test (VWF:RCo)
– HemosIL von Willebrand Factor Activity Assay
Confirmatory Lab Tests
 VWF:RCo
– Ristocetin promotes binding of vWF and
platelet glycoprotein Ib
– Measures amount and rate of platelet
aggregation
– Challenges
• Macroscopic slide method is semi-quantitative
• Platelet aggregometer is labor intensive
• High inter-assay and inter-laboratory variability
Confirmatory Lab Tests
 HemosIL von Willebrand Factor Activity
assay
– Monoclonal antibody binds to platelet-binding
site of vWF (glycoprotein Ib receptor)
– Antibody is absorbed onto latex
– Test measures turbidity that results from
agglutination of latex
The Question
P
Patients suspected of von Willebrand
Disease
I
C
O
Automated assay for vWF activity
Ristocetin co-factor test
Detecting and classifying vWD
Are automated assays done on patients suspected of von
Willebrand Disease more or as accurate in the diagnosis of the
disease compared with the ristocetin co-factor test?
Search Strategy
 PubMed Clinical Queries, accessed
through the USF Shimberg Library
– Reliable, open database
– Provides clinically relevant information that can
help answer case questions
 Search “von Willebrand factor AND
Ristocetin AND automated”
 Found a cross-sectional study
– Abstract included details of interest and study
objectives paralleled those of scenario
Critical Appraisal
Objectives
 Are the objectives of the study clearly
stated?
– Yes
Design
 Is the study design suitable for the
objectives?
– Yes
– The study was a prospective cross-sectional
study, which is the best study design for
assessing diagnostic tests.
• Advantages: simple, ethically safe, relatively cheap
• Disadvantages: establishes association, recall bias,
confounders, unequal group sizes
Design
 Who/what was studied?
Design
 Was this the right sample to answer the
objectives?
– Yes
– The authors selected a sample that avoids spectrum
bias
• All were suspected to have vWD; none were obviously
diseased or obviously healthy
– The patients were recruited consecutively indicating
that selection bias is less likely.
– Several blood types were represented. However, only
two types of vWD were represented, indicating the full
spectrum of vWD patients were not represented.
Design
 Is the study large enough to achieve its
objectives? Have sample size estimates
been performed?
– Uncertain
• There are no indications that a power analysis was
performed.
• The sample size was relatively small (54 samples
had complete data), but this is acceptable because
vWD is relatively rare.
• The authors admit that studies with more patients
need to be done.
Design
 Were all subjects accounted for?
– Yes
– They did not provide all the individual sample data but
the tables all add up.
Design
 Were all appropriate outcomes considered?
– Yes
– The authors considered all possible test result
combinations
 Has ethical approval been obtained if
appropriate?
Design
 Was an independent blinded gold standard
test applied to all subjects?
– The gold standard test, ristocetin cofactor
assay, was performed on all subjects.
– It does not seem to have been independent
• The authors performed both the gold standard and
the test assay
– It is unclear if the researchers were blinded,
but considering the objectivity of the measures
being compared, this may not be very critical.
Measurement
 Is it clear what was measured, how it was
measured, and what the outcomes were?
– Agglutination of latex reagent at 405 nm
– Decrease in light transmittance
– Reference curves were utilized
– MDA-180 & ACL TOP coagulation analyzers
were utilized
Measurement
 Are the measurements valid?
Measurement
 Are the measurements reliable?
– No conspicuous discrepancies exist amongst
samples and equipment
Measurement
 Are the measurements reproducible?
– Not well described
– They are likely to be reproduced due to quality
control tests from the manufacturers, no
significant differences between coagulation
analyzers, and standard manufacturers’
protocol (and dilutions) applied to all assays
Results
 Are the basic data adequately described?
– Yes
– Percentage agglutinations of each sample are
clearly grouped and explained as to whether or
not vWD is associated and, if so, which type
Results
 Are the results presented clearly,
objectively, and in sufficient detail to enable
readers to make their own judgment?
– Yes
Results
 Are the results internally consistent, i.e. do
the numbers add up properly?
– Yes
– Numerators and denominators included for
most percentage calculations
– Table 2 (previous slide) provided to show
exactly how all cases were classified
Analysis
 Are the data suitable for analysis?
– Yes
– Sensitivity and specificity desired for diagnostic
study analyses
Analysis
 Are the methods
appropriate to the
data?
– Yes
– For the type of
diagnostic analysis
needed, the
agglutination
methods are
appropriate
Analysis
 Are any statistics correctly performed and
interpreted?
– Hard to tell because no clear chart or individual
patient data is provided
– However, from the Table 2, we can calculate:
• Specificity= 36/42= 86%
• Sensitivity= 12/12= 100%
– Positive predictive and negative predictive
values provided because the true spectrum of
patient population is not represented
Discussion
 Are the results discussed in relation to
existing knowledge and on the subject and
study objectives?
– Yes
– The results explicitly mention the current gold standard of
testing and the relation of the new method.
– They briefly mention the relation of vWD and blood types, Factor
VIII activity and PTT as well.
– Furthermore, the results are provided as evidence to support the
study objective, which was to analyze a new, quicker, easier test
to screen for (and ultimately diagnose) vWD that could mitigate
the reliance upon more complex tests, such as the Ristocetin
Cofactor tests, to potentially save time and money.
Discussion
 Is the discussion biased?
– The discussion does not appear to be biased,
but it is unknown if the researchers have a
conflict of interest
– They clearly favored the implementation of the
new test, however, the data supported their
claims of quality.
Interpretation
 Are the authors’ conclusions justified by the
data?
– Yes
– The data had 100% sensitivity and over 85%
specificity, which supports the claim that the
new test is a viable screening alternative.
– However, the study was small, there was still
sufficient heterogeneity, and all types of results
were found, including normal.
Interpretation
 What level of evidence has this paper
presented (using CEBM levels)?
– Level 1b or 1c
• Study was a “validating” (not exploratory) study
• Had good reference standard application
• Absolute SnNout (sensitivity=100%)
– Grade A recommendation
• consistent with a level 1 study
OCEBM Levels of Evidence Working Group. "The Oxford 2009 Levels of Evidence". Oxford Centre
for Evidence-Based Medicine. http://www.cebm.net/?o=1025
Interpretation
 Does this paper help me answer my problem?
– Yes, it does, at least preliminarily.
– I can utilize this new test with my patient (as long as it
is cost-efficient) to rule out vWD. However, if the
results are abnormal, I will have to retest utilizing the
gold standard of the Ristocetin Cofactor test to be
certain.
 How do you rate this paper? 8
– It seems to provide useful information, but with a
limited patient population and data set, I cannot be
certain of the validity of the results
Case Review
 A 21 year old woman presents to your office for evaluation of
abnormal menstrual periods. Her periods last from 7 to 9 days
requiring up to 10 pads a day (excessive). On further
questioning, she admits to easy bruising and nosebleeds.
Family history reveals that her mother has had similar
problems.
 You suspect von Willebrand disease (vWD). vWD is the
most common inherited coagulation abnormality.
 Your laboratory informs you of a new automated assay for
vW factor (vWF) activity. Since you have not heard about it,
you decide to check what the published literature says about
this new test and how does it compare to the established
ristocetin cofactor method that you used to order to confirm
the diagnosis of vWD.
Diagnosis of von Willebrand Disease
 Three main criteria:
– Past medical history of excessive bleeding
• Periods last from 7 to 9 days, requiring up to 10
pads per day
• Easy bruising
• Nosebleeds
– Family history of excessive bleeding
• Mother has had similar problems
– Laboratory evaluation of vWF
Diagnosis of von Willebrand Disease
 Laboratory
evaluation of
vWF
– Follow Figure 2
to determine
procedure for
ordering tests:
Implementation
 Can any necessary change be implanted in
practice?
– Yes
– As long as the new test is readily available and
cost-effective, it would likely be a favored
alternative
Implementation
 What aids to implementation exist?
– Technology, marketing, and coding. Since technology
is so advanced, it should be relatively easy to order the
required supplies and have the tests available “inhouse” in a short period of time.
– With marketing, more people could be informed about
the new, “easy” test and opt to have it done, especially
if it is faster, cheaper, and easier than the old method.
– Depending on the medical coding being used in each
clinic or hospital, a new code could be created to bill
the patient or the patient’s insurance for the test.
Implementation
 What barriers to implementation exist?
– Insurance companies might be reluctant to pay for new
technology, especially if it implicates further disease
states that it will be responsible for covering.
– Some physicians and/or facilities may not want to try
something new, especially if they are used to the “old
ways” of doing things.
– Depending on how much time and money is really
saved by the new method, insurance companies may
not want to cover it, especially if the patients end up
having a second test done as well.
Implementation
 Are my patients the same as the patients
tested?
– Yes, approximately.
– The mean age was 37 (range 2-89 years), and
my patient is 21, so she falls within the range
of patients tested.
Implementation
 Will the test improve diagnosis in my
patients?
– It may not improve the diagnosis per se,
however, it may make the process easier and
completed more quickly and easily, which
could potentially correlate to savings of money
and time.
– Therefore, if I had several patients who needed
to be tested, it might improve their diagnosis in
that they get it more quickly and thus receive
proper treatment more quickly as well.
References
1. Salem, RO, Van Cott, EM. A new automated screening
assay for the diagnosis of von Willebrand disease. Am J
Clin Pathol 2007; 127:730-735.
2. De Vleeschauwer, A, Devreese, K. Comparison of a new
automated von Willebrand factor activity assay with an
aggregation von Willebrand ristocetin cofactor activity
assay for the diagnosis of von Willebrand disease. Blood
Coagul Fibrinolysis 2006; 17:353-358.