From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., "Coagulation." In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.
Index of Tests
von Willebrand Factor [CO006200]
Synonyms Multimer Assay; Ristocetin Cofactor; Ristocetin-Induced
Platelet Aggregation Assay; von Willebrand Factor Antigen; von Willebrand
Factor Assay; von Willebrand Factor Collagen-Binding Assay; von
Willebrand Factor Multimer Assay
Applies to Acute Phase Reactant; DDAVP; Desmopressin; Factor
VIII:von Willebrand Factor Ratio
Test Includes Assays for von Willebrand factor (vWF) activity
(ristocetin cofactor), vWF antigen, and factor VIII should be ordered.
If indicated by these results, a vWF multimer analysis and/or low-dose
ristocetin aggregation assay can be ordered.
Abstract von Willebrand factor (vWF) mediates platelet adhesion
to injured endothelium, the first step in hemostasis. It also helps
maintain factor VIII levels. When vWF is deficient, patients have
a bleeding disorder called von Willebrand disease (vWD). vWD is
the most common hereditary bleeding disorder, of which several subtypes
are recognized (see below).
Container Three blue top (sodium citrate) tubes
Collection Routine venipuncture. Deliver tubes to laboratory
immediately, otherwise falsely low factor VIII values may occur
(factor VIII is labile). If multiple tests are being drawn, draw
blue top tubes after any red top tubes but before any lavender top
(EDTA), green top (heparin), or gray top (oxalate/fluoride) tubes.
Immediately invert tubes gently at least 4 times to mix. Tubes must
be appropriately filled.
Storage Instructions Separate plasma from cells as soon as
possible. Plasma can be stored for 2 hours on ice, otherwise store
frozen. For vWF antigen only, plasma can be stored for 8 hours at
room temperature or 24 hours on ice, otherwise store frozen.
Causes for Rejection Specimen received more than 4 hours
after collection, tubes not filled, clotted specimens
Turnaround Time Several days (longer if follow-up testing
is needed, such as multimer analysis)
Reference Interval Varies with blood type through an unknown
mechanism. Results are reported as a percent of the amount expected
in normal plasma. By definition, the mean value in pooled normal
plasma is 100%. In a large study of normal persons, the mean vWF
level was 75% in blood type O, 106% in type A, 117% in type B, and
123% in type AB individuals. The overall mean vWF level was 100%.1 Newborns have higher vWF levels than do adults. Values for vWF gradually
decrease into the adult normal range by age 6 months.2
Use Determine if a patient with a personal or family history
of bleeding has von Willebrand disease (vWD); assist in determining
hemophilia A carrier status in females
The ristocetin cofactor assay assesses vWF function by measuring
ristocetin-mediated binding of vWF to platelet GPIb, which leads
to platelet agglutination.3 The ristocetin cofactor assay
is performed by mixing patient plasma with ristocetin and formalin-fixed
normal platelets and measuring the amount of platelet agglutination
in an aggregometer. Note: The term "agglutination" is often
used to describe ristocetin-induced platelet aggregation, because
true platelet aggregation links platelets through fibrinogen and
GPIIb/IIIa, whereas ristocetin links platelets through von Willebrand
factor and GPIb. The von Willebrand factor antigen assay
is an enzyme-linked immunosorbent assay (ELISA), which measures
the quantity of vWF, independent of vWF function. An alternative
automated assay involves latex particles coated with antibodies
directed against vWF. In the presence of vWF, the latex particles
form aggregates that absorb light passing through the specimen.
The amount of light absorbance is directly related to the amount
of vWF in the specimen. Rocket immunoelectrophoresis is an older
antigen assay that is still in use in some laboratories. The factor
VIII assay is a PTT-based clotting assay which measures factor
More recently, alternative immunoassays have been designed to assess
vWF function.4 The collagen-binding assay is an ELISA
in which collagen is the antigen. If vWF is functional, it binds
to collagen and is subsequently detected. Another vWF "functional"
ELISA uses monoclonal antibodies that recognize a functional epitope
on vWF, but there is conflicting evidence whether this test correlates
better with ristocetin cofactor or with vWF antigen. These newer
functional assays are not yet as established as the ristocetin cofactor
Follow-up tests (performed if indicated, see table):
Multimer analysis is performed when type 2 vWD is suspected.5 A plasma sample is electrophoresed on a gel to separate the multimers
by size. The multimers are then visualized using 125I-labeled
anti-vWF antibody or other techniques. Low-dose ristocetin platelet
aggregation assay is performed when type 2B vWD is suspected.6 This test is similar to the ristocetin cofactor assay, except that
the patient's own platelets are used instead of normal platelets,
and lower doses of ristocetin are used. The patient's own platelets
and plasma are mixed with ristocetin, and platelet aggregation is
measured in an aggregometer. This assay is less sensitive than the
ristocetin cofactor assay for diagnosing vWD, but it is useful for
confirming a diagnosis of type 2B vWD. Type 2B patients' platelets
become abnormally coated with vWF in vivo, due to increased
affinity of the mutant vWF for platelet GPIb. As a result, the patient's
platelets show increased aggregation in this assay. Platelet-type
vWD also shows increased aggregation in this assay, due to a mutation
on platelet GPIb which increases its affinity for vWF. In contrast,
other types of vWD may show decreased ristocetin-induced platelet
aggregation due to decreased vWF quantity and/or function. Further
specialized coagulation testing can be performed to distinguish
type 2B from platelet-type vWD.
Additional Information von Willebrand disease (vWD) is the
most common hereditary bleeding disorder, occurring in up to 1%
of the general population.7,8 Many cases remain undiagnosed
because of the mild nature of bleeding in many patients and the
fact that acute phase reactions can mask the diagnosis. vWF is a
polypeptide synthesized in endothelial cells and megakaryocytes,
which polymerizes to form multimers containing up to 100 subunits.
Bleeding symptoms resemble those of a platelet function defect,
since platelet adhesion is impaired. Thus, the most common symptoms
are epistaxis, easy bruising, bleeding with dental extractions,
Laboratory testing for vWD is summarized in the table. Repeat testing
is often required, because both vWF and factor VIII become elevated
above baseline during acute phase reactions (including even minor
illnesses, injury, or stress), pregnancy, estrogen use, or in newborns. An elevation of a low or borderline value for vWF into the normal
range during any of these conditions often masks the diagnosis of
vWD. Measurement of an acute phase reactant such as fibrinogen
is helpful in assessing the likelihood that a patient is in an acute
phase reaction at the time of testing.
vWF serves as the carrier protein for factor VIII, and levels of
factor VIII are often decreased when vWF is decreased. When vWF
is markedly decreased, the factor VIII level can also become very
low, which prolongs the PTT. In most vWD patients, the disease is
mild or moderate and the PTT is therefore normal.
Many variants of vWD have been described, but the classification
scheme has recently been simplified into three types (see table).9 Type 1 is by far the most common form, accounting for the majority
of cases. Type 1 vWD is characterized by a partial quantitative
deficiency of vWF. Although the quantity of vWF is reduced, the
function of the individual vWF molecules which are synthesized is
Interpretation of von Willebrand Factor Assays
RCoF + vWF Ag + FVIII + Fibrinogen
(or other acute phase reaction marker):
* Normal:* vWD unlikely if no acute phase reaction, pregnancy,
estrogen use, newborn
* Normal* but fibrinogen (or factor VIII) elevated: repeat
vWF assays when fibrinogen and factor VIII are normal
* RCoF, vWF Ag, FVIII reduced to a similar extent: type
1 vWD likely
* RCoF, vWF Ag, FVIII severely reduced (<10%) or undetectable: type 3 vWD likely
* RCoF reduced more severely than vWF Ag and FVIII:** consider type 2 vWD (2A, 2B, or 2M); perform multimer analysis
and low-dose ristocetin cofactor to determine subtype:
- Multimer analysis normal: type 2M likely (subtle abnormalities
in some variants)
- Multimer analysis missing high molecular weight multimers:
type 2A likely
- Multimer analysis missing high and intermediate molecular
weight multimers: type 2B or platelet type likely
- Increased low-dose ristocetin aggregation: type 2B or platelet
- Normal or decreased low-dose ristocetin aggregation: not
type 2B or platelet type
* FVIII reduced (5% to 40%), RCoF and vWF Ag normal:** consider type 2N vWD; or in males, mild hemophilia A. In female
hemophilia A carriers, factor VIII is approximately 50% with
large variability. Consider also factor VIII degradation if
prolonged specimen transportation.
*Consider blood type when determining if values are normal.
**Mean RCoF:vWF Ag ratio is 0.3 for type 2A, 0.6 for type
2B, and uncertain (<1) for type 2M. Mean FVIII:vWF Ag ratio
is 0.28 for type 2N (see Footnote 13).
***Thrombocytopenia may occur with type 2B or platelet-type
(and rare type 2A variants).
RCoF = ristocetin cofactor assay; vWF Ag = von Willebrand
factor antigen assay; FVIII = factor VIII assay
Type 2 vWD is characterized by qualitative (functional) deficiencies
of vWF. Often the quantity of vWF is also reduced. Type 2 vWD is
further subdivided into four categories (see table). Type 2A and
type 2B are characterized by a loss of high molecular weight multimers
of vWF. The highest molecular weight multimers have more hemostatic
function than the lower molecular weight multimers. Therefore, in
these disorders, the overall function relative to the quantity of
vWF molecules is reduced. Thus, the functional assay (ristocetin
cofactor) result is reduced more than the quantitative assay (von
Willebrand factor antigen). In type 2A, the loss of high molecular
weight multimers is due to defective multimer assembly and secretion
or increased proteolysis of multimers.10,11
Type 2B vWD mutations lead to increased binding of vWF to GPIb,
the platelet vWF receptor.6 Platelets coated with vWF
are cleared from the bloodstream at an increased rate, leading to
loss of high molecular weight multimers as well as thrombocytopenia.
Platelet-type or pseudo-vWD is a rare disorder in which a mutation
in the platelet GPIb gene (not the vWF gene) leads to increased
binding of vWF to GPIb, resulting in the same findings described
above for type 2B vWD.12
Types 2M and 2N vWD are rare subtypes of type 2 vWD. Type 2M vWF
mutations cause decreased function despite the presence of normal-sized
multimers, often because the mutation impairs the ability of vWF
to bind to platelet GPIb.9,13 In type 2N (Normandy) vWD,
the factor VIII-binding ability of vWF is impaired, and the half-life
of factor VIII is consequently shortened. Thus, vWF is normal in
quantity (normal antigen assay) and has normal platelet-adhesion
function (normal ristocetin cofactor assay), but factor VIII levels
are decreased. As a result, type 2N patients are frequently misdiagnosed
as having hemophilia A.14 The family history may
be useful in distinguishing type 2N vWD from hemophilia A. Type
2N vWD is inherited autosomally (males and females are affected),
whereas hemophilia A is an X-linked recessive disorder (males are
affected and females are carriers). An assay which measures the
ability of vWF to bind factor VIII is available in a limited number
of specialized laboratories. Type 2N patients show decreased binding
of factor VIII in this assay.
Type 3 vWD is a rare, severe bleeding disorder characterized by
a severe quantitative deficiency of vWF such that vWF is typically
The bleeding time is often prolonged in vWD. However, it is neither
a necessary nor a reliable test for diagnosis.
In hemophilia A carriers (who are females only), the factor VIII:vWF
ratio is ~0.5, instead of the normal ratio of 1. Definitive determination
of carrier status may require DNA-based testing for mutations that
cause hemophilia A.
Bleeding episodes, in most patients, can be treated with DDAVP
(desmopressin) if needed, as DDAVP temporarily increases the levels
of vWF and factor VIII two- to threefold. As a small percentage
of patients do not respond to DDAVP, patients are usually given
a trial dose of DDAVP while asymptomatic, with measurement of their
vWF level before and after DDAVP, to ensure that their vWF levels
do increase with DDAVP. Bleeding patients who do not respond to
DDAVP or patients with severe vWD can be treated with vWF-containing
factor VIII concentrates (eg, Humate-P®, Alphanate®, Koate®).
Some consider DDAVP contraindicated in type 2B because it can cause
thrombocytopenia. However, others report DDAVP is a beneficial treatment
for type 2B patients.
Acquired vWD is a rare condition that can occur spontaneously or
in association with a variety of underlying disorders, such as hematologic
neoplasms or autoimmune diseases. Thrombotic thrombocytopenic purpura
(TTP) is due to a deficiency of a vWF-cleaving protease, usually
due to an autoantibody against the protease.15,16 This
could account for the microvascular platelet-rich thrombi and thrombocytopenia
that are characteristic of TTP. Unusually large vWF multimers may
also be seen in TTP.
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Internet Web Sites
mmg2.im.med.umich.edu/vwf (database of vWF mutations)