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2172 Part XII: Hemostasis and Thrombosis Chapter 126: von Willebrand Disease 2173

VON WILLEBRAND FACTOR ANTIGEN migrating more slowly than the intermediate or smaller multimers.
Plasma VWF:Ag is usually quantitated by electroimmunoassay or an The multimers may be visualized by autoradiography after incubation
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enzyme-linked immunosorbent assay (ELISA) technique. In type 1 with I-monospecific antihuman VWF antibody or, more commonly,
VWD, the VWF:Ag assay usually parallels VWF:RCo, but it has lower by nonradioactive immunologic techniques. The normal multimeric
specificity and sensitivity than the VWF:RCo assay. In patients with distribution is an orderly ladder of major protein bands of increasing
type 2 VWD, the VWF:Ag is variably decreased but can be normal molecular weight, going from the smallest to the largest VWF multim-
(see Table 126–2). ers (see Fig. 126–4). Each normal multimer has a fine structure consist-
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ing of one major component and two to four satellite bands. Type 2B
and most of the type 2A variants were initially distinguished from each
RISTOCETIN COFACTOR ACTIVITY other on the basis of subtle variations in the satellite band pattern. In a
The standard measure of VWF activity, the VWF:RCo quantitates the large European multicenter type 1 VWD study, careful analysis of VWF
ability of plasma VWF to agglutinate platelets via platelet membrane multimers in subjects historically diagnosed as type 1 VWD, including
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GPIbα in the presence of ristocetin. In the most common method, patients diagnosed at experienced centers, found one-third of “type 1”
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normal platelets washed free of plasma VWF are used either as fresh VWD patients had subtly abnormal multimers. Although this pre-
platelets or after formaldehyde fixation. This assay has long been viously would have required reclassification of these patients as type
reported to be the most sensitive and specific single test for the detec- 2 VWD, the most recent update on the classification of VWD by the
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tion of VWD. Numerous alternative methods have been proposed International Society on Thrombosis and Haemostasis Subcommittee
as adjuncts or replacements of the standard platelet-based ristocetin on von Willebrand Factor expanded the category of type 1 VWD to
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cofactor activity assay. However, none as yet can serve as a surrogate for permit subtle VWF multimer abnormalities. The authors of the Euro-
VWF:RCo (reviewed in Ref. 290). pean type 1 VWD study note that having samples from the index case,
Ristocetin cofactor activity is generally decreased coordinately affected family members, and unaffected family members on one gel
with VWF:Ag and FVIII in type 1 VWD patients. In type 2 VWD vari- made qualitative defects more readily detectable, and that intermedi-
ants, ristocetin cofactor activity can be disproportionately decreased, ate-resolution multimer gels were superior to low-resolution multimer
as is usually the case in type 2A variants (and sometimes type 2B), gels in detecting abnormalities in this population. Use of VWF tests sen-
because of the greater dependence of the ristocetin-mediated platelet– sitive to VWF multimer structure have been proposed by some experts
VWF interaction on the presence of larger VWF multimers, and in type as proxies for VWF multimer testing, such the VWF:RCo-to-VWF:Ag
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2M, because of decreased VWF-platelet interactions (see Table 126–2). ratio (VWF RCo:Ag ratio) or VWF:CB assay. In studies comparing
Thus, the VWF:RCo-to-VWF:Ag ratio has been proposed as a means these approaches as surrogates for VWF multimer assays, the (VWF
to distinguish between type 1 and type 2 VWD, with a ratio of VWF:R- RCo:Ag ratio) ratio was found to be less sensitive than multimer gel
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Co-to-VWF:Ag of less than 0.7 being indicative of a qualitative (type 2) techniques in identifying qualitative VWF defects, while VWF:CB
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VWF defect. However, in patients with very low VWF:Ag levels this an detect some type 2M VWD patients who have normal VWF
ratio may not be reliable because of the limits of sensitivity of most multimers. 294,295 These observations support a continued important role
VWF:RCo assays. The VWF:RCo assay is uninformative in the pres- for VWF multimer analysis in the laboratory evaluation of VWD.
ence of the VWF Asp1472His variant, which interferes with the VWF–
ristocetin interaction in vitro. 155 ADDITIONAL LABORATORY TESTS
As a result of the variable sensitivity and specificity of laboratory testing
RISTOCETIN-INDUCED PLATELET for VWD, additional diagnostic studies may be useful in the classifica-
AGGLUTINATION tion of VWD patients. The VWF:CB measures VWF binding to col-
lagen (type I, type III, type VI, or mixed) by ELISA. As above, assays
Similar to the ristocetin cofactor assay above, the RIPA assay also based upon VWF:CB can complement the VWF:RCo in detecting type 2
measures platelet agglutination caused by ristocetin-mediated VWF VWD variants, 296–299 and an abnormal VWF:CB-to-VWF:Ag ratio (VWF
binding to platelet membrane GPIbα. In the case of RIPA, ristocetin is CB:Ag ratio) is suggestive of a qualitative VWF defect. Abnormalities
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added directly to patient platelet-rich plasma and platelet aggregation in VWF:CB can reflect loss of high-molecular-weight multimers and/
is measured. Hyperresponsiveness to RIPA results either from a type or the discrete loss of collagen binding caused by a type 2M mutation.
2B VWD mutation or an intrinsic defect in the platelet (platelet-type or Use of VWF:CB assays is expanding in clinical practice, with select sites
pseudo-VWD). In these disorders, patient platelet-rich plasma aggluti- including this test routinely in initial VWD diagnostic laboratory testing.
nates spontaneously or at low ristocetin concentrations of only 0.2 to 0.7 Another group of tests are included in the term VWF “activity”
mg/mL. At these concentrations, normal platelet-rich plasma does not (VWF:Act). These tests seek to assess VWF-GPIb binding capacity
agglutinate. Type 2B and platelet-type VWD can be distinguished by independent of ristocetin, usually by using antibodies to the VWF A1
RIPA experiments performed with separated patient platelets or plasma domain in ELISAs. These tests are easily confused with the VWF:RCo,
mixed with the corresponding component from a normal individual which has also been referred to as “VWF activity.” None of these tests
or paraformaldehyde-fixed platelets. The RIPA is generally reduced in measures activity; rather both VWF:RCo and VWF:Act are sensitive to
most other subtypes of VWD (see Table 126–2). VWF conformation. The VWF:Act does not always provide the same
results as VWF:RCo, particularly with regards to type 2M VWD, and
is not considered a substitute for the VWF:RCo (reviewed in Ref. 290).
MULTIMER ANALYSIS When type 2N VWD is suspected, VWF:FVIII binding capac-
Analysis of plasma VWF multimers is critical for the proper diagnosis ity can be measured. Specific assays of FVIII binding to VWF
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and subclassification of VWD (see Fig. 126–4). This is generally accom- (VWF:FVIIIB) have been developed and can be used to confirm the
plished by agarose gel electrophoresis of plasma VWF to separate VWF diagnosis of type 2N VWD. 300,301 Type 2N carriers do not always exhibit
multimers on the basis of molecular size, with the largest multimers a decrease in VWF:FVIIIB, but a decreased VWF:FVIIIB-to-VWF:Ag

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