COAGULATION AND TRANSFUSION MEDICINE
Original Article
Laboratory Assessment of
von Willebrand Factor
Use of Different Assays Can Influence the Diagnosis of von
Willebrand's Disease, Dependent on Differing Sensitivity
to Sample Preparation and Differential Recognition
of High Molecular Weight VWF Forms
EMMANUEL J. FAVALORO, PHD, DAVID FACEY, BSC(HONS), AND LINDA GRISPO
Three separate laboratory assays for von Willebrand Factor (VWF), a
standard "antigen" (antisera-ELISA-based) assay (VWF:Ag), a standard ristocetin-dependent-platelet-agglutination procedure (VWF:
RCof), and an ELISA-based collagen-VWF binding assay (VWF:
CBA), have been evaluated for their ability to detect alterations in VWF
levels following differential processing of blood for testing, and specifically in (1) serum compared to plasma and (2) filtered plasma compared
to nonfiltered plasma. Although all assays tended to detect some
change, sensitivity of detection varied between assays, with the VWF:
CBA most consistently able to detect large decreases in VWF levels in
serum and filtered plasma.
The authors propose that the increased sensitivity of the VWF:CBA
assay to VWF depleted in these circumstances is that this assay selectively detects higher molecular weight forms (ie, those known to be
more functionally relevant), and that assay results reflect the preferential incorporation of these forms in the platelet-fibrin-gel during the
clotting process, and onto the filter matrix during filtration. To confirm
this, multimer analysis was performed and showed a reduction in high
molecular weight forms of VWF in these cases. Finally, direct evidence
that the VWF:CBA assay preferentially detects high molecular weight
forms of VWF was obtained following fractionation of normal plasma
VWF (separation according to molecular weight using size exclusion
matrix; confirmed by specific multimer analysis) and assessment of
eluted VWF. Using a standard VWF:Ag assay, detection of eluted
VWF was unrelated to molecular size. In contrast, the VWF:CBA
showed selective detection, and was able to preferentially discriminate
high and intermediate forms of VWF from low molecular weight forms.
The findings are of particular relevance to diagnostic pathology laboratories because filtered plasma or serum can be inappropriately (and unknowingly) provided for the clinically queried diagnosis of von Willebrand's disease (VWD). As outlined in this report, these samples can
yield VWF results that closely mimic those of a Type 2A or Type 2B
VWD individual, and thus, VWD may be incorrectly diagnosed. (Key
words: von Willebrand factor; von Willebrand's disease; Plasma; Serum; Collagen) Am J Clin Pathol 1995; 104:264-271.
von Willebrand factor (VWF) is an adhesive plasma protein with two primary hemostatic roles, permitting adhesion of platelets to sites of vascular damage, and acting
to stabilize factor VIII coagulation function.1"4 Patients
diagnosed as having von Willebrand's disease (VWD),
which is currently recognized as the most common inherited bleeding disorder,4 suffer defects in, or have reduced levels of, VWF, and are typed according to classi-
fication protocols involving both clinical observations
and laboratory analysis.1"6 Typically, laboratory investigation involves a battery of tests, including determination of (skin) bleeding times, VWF antigen (VWF:Ag)
levels, "functional" activities, such as factor VIII coagulant and Ristocetin cofactor (VWF: RCof) levels, and
VWF multimeric analysis.1'2 Classification of a patient's
VWD is also important, not only because the biologic
activity of VWF is related to its multimer profile, but
because subsequent clinical management of such patients may differ substantially on this basis.7'8
To improve and simplify our laboratory's testing procedure for diagnosis and classification of VWD, we refined the original collagen-based ELISA assay for VWF
described by Brown and Bosak9 to develop an assay capable of (1) sensitive discrimination between plasma derived from Type 2 (subtypes 2A and 2B) VWD patients,
From the Haemostasis and Thrombosis Research Unit, Diagnostic
Haemostasis Laboratories, Department of Haematology, Institute of
Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Westmead, Australia.
Manuscript received November 15, 1994; revision accepted April 19,
1995.
Address reprint requests to Dr. Favaloro: Department of Haematology, ICPMR, Westmead Hospital, Westmead, NSW 2145, Australia.
264
FAVALORO, FACEY, AND GRISPO
Collagen Binding Assay for VWF
and that derived from either Type 1 VWD patients, or
from normal individuals; 10 " and (2) detection of some
VWD patients otherwise missed by a more restricted
screening protocol." More recently, we showed that this
assay might also provide a more sensitive marker of true
functional VWF responsiveness to desmopressin
(DDAVP) therapy in patients with VWD. 12 Although
our assertion has been that the unique power of the
VWF:CBA stems from its ability to preferentially bind
high molecular weight (ie, more functionally relevant)
forms of VWF, this had never been formally assessed.
Accordingly, the characteristics of this assay have been
investigated in more detail in this study, which provides
direct evidence that the VWF:CBA possesses unique
high sensitivity to larger molecular weight forms of
VWF. Furthermore, the VWF:CBA assay has enhanced
ability to detect differences in VWF between plasma and
serum, and in plasma following nitration. In addition to
the important physiologic implications, these findings
suggest caution in the interpretation of abnormal VWF
data derived in a diagnostic pathology laboratory that
may follow testing in unknowingly received (and inappropriate) serum or filtered plasma samples, and on the
basis of data outlined in this report, which could subsequently lead to an incorrect diagnosis of VWD.
MATERIALS AND METHODS
Blood Samples
These were derived from normal healthy volunteers
using venous blood collected into standard blood collection tubes (Becton Dickinson, Rutherford, NJ) containing either no additive (glass tube, no separator, blood left
undisturbed at 22 °C for 1 to 2 hours to allow it to clot),
or 0.105 M buffered sodium citrate (9 parts blood, 1 part
citrate; blood processed immediately), and subsequently
centrifuged (2,000 g; 15 minutes) to remove cellular
components to yield serum and citrated plasma samples,
respectively. For some experiments, plasmas obtained
from individual normal donors (IND) were filtered
through a standard 0.22 » filter device (MILLEX-GS filters, Millipore, Sydney, Australia). In addition to individual assessment, unfiltered individual citrated plasmas
were also combined to create a large pool (>80 normal
individuals; PNP) for assay calibration purposes. All
plasma and serum samples collected were snap frozen
in aliquots (at - 7 0 °C, chest freezer) to permit multiple
analysis by various assays.
Assays for von Willebrand Factor
These comprised assays for VWF:multimer analysis,
von Willebrand factor antigen levels (VWF:Ag), Risto-
265
cetin cofactor (VWF:RCof) functional activity, and a
"functional" collagen binding assay (VWF:CBA) as described subsequently, and elsewhere.10-1'
ELISA Assay for YWF-.Ag
This assay, a standard sandwich indirect 96-well procedure, has been described in detail elsewhere. 10 "
Briefly, 96-well plates (EIA plates, ICN-Flow, Australia)
were allowed to coat with titered concentration of rabbit
antisera raised to human VWF (isolated immunoglobulin fraction; Dakopatts, Denmark; overnight, 4 °C in a
humidified container; antisera typically used at 1:1,000
final dilution, 200 /xh per well, in 0.1M N a H C 0 3 buffer,
pH 8.3). Following washing (X3) in ELISA buffer (0.12
M NaCl, 0.02 M imidazole, 0.005 M citric acid, 0.1%
BSA; pH 7.3), plates were incubated with 5% BSA (in
ELISA buffer) to reduce nonspecific background binding
(1 hour; 22 °C). Plates were rewashed (X3), and then allowed to incubate with a suitable dilution of individual
patient plasma or serum (typically 1:100 final dilution
in ELISA buffer), in triplicate wells, 200 uL per well. A
calibration curve was always set up using PNP (see references 10, 11). After 2 hours, 22 °C, plates were rewashed
(X4), and then allowed to incubate (2 hours; 22 °C) with
a suitable (pre-titered) concentration of horseradish peroxidase (HRP) labelled rabbit antisera raised to human
VWF (Immunoglobulin fraction; Dakopatts, Denmark;
typically 1:1,000 final concentration in ELISA buffer,
200 /iL per well). Plates were rewashed (X5), and allowed
to incubate with peroxidase substrate (TMB; Tetramethyl benzidine HC1; Sigma Chemical, St. Louis, MO;
final concentration 0.42 mM in 0.1 M Na acetate/citric
acid buffer, pH 6.0, with 0.3mM H 2 0 2 added) to permit
color development. The reaction was stopped when required (typically after 5 minutes) by addition of 50 /*L
per well of 2M H 2 S0 4 . Absorbances were read (at 450
nm against 550 nm) using an automated plate reader (Biotek Instruments, model 312, Winooski, VT) incorporating a data management software package. For the purpose of this report, test plasma or serum values for VWF:
Ag have been calculated with reference to PNP (= 1 U
VWF/mL).
Ristocetin Cofactor (VWF:RCof) Assay
This assay has also been described in detail elsewhere, 1 0 " and uses platelet agglutination. Briefly, normal donor platelets were washed twice in isotonic phosphate buffered saline (PBS) containing 5mM EDTA, and
then fixed (10 minutes, 22 °C) using 1% paraformaldehyde (final concentration) in PBS/EDTA. Platelets were
Vol. 104.No. 3
266
COAGULATION AND TRANSFUSION MEDICINE
Original Article
again washed (X2) in PBS/EDTA, and then resuspended
in PBS (no EDTA) at a concentration of 250 X 106/mL
for use in the assay. Ristocetin cofactor activity was then
determined using platelet agglutination (Paytons Associates aggregometer, model 1000, Scarborough, Canada).
To 400 nL of platelets was added 50 nL of plasma, or
dilution, followed by 50 fiL of ristocetin (Wellcome,
Australia; 1 mg/mL final assay concentration). Platelet
agglutination was monitored, and a calibration curve
(using the slope of the initial agglutination response) was
generated from PNP (tested at least three times using
four different concentrations; undiluted, 1:2, 1:4, 1:8
with PBS); test plasma or serum was typically tested at
three concentrations (undiluted, 1:2, 1:4, with PBS). For
the purpose of this report, test plasma or serum values
for VWF:RCof were calculated with reference to PNP (=
1 U VWF/mL).
Collagen Binding Assay for VWF (VWF.CBA)
This assay, together with a detailed analysis of assay
variables, has been outlined previously.10,1' It is based on
modifications to the assay described for VWF:Ag above,
and the collagen-VWF assay originally described by
Brown and Bosak.9 Unless otherwise stated, solutions
and materials used are identical to the assay for VWF:
Ag previously described. In the collagen binding assay
(VWF:CBA), new 96-well plates are allowed to coat with
collagen (ie, instead of rabbit immunoglobulin raised to
VWF). A number ofdifferent collagen preparations and
conditions have so far been evaluated by our laboratory, 10 " and these give rise to differences in the ability
of the assay to preferentially recognize high molecular
weight VWF forms. It is thus important to select a suitable collagen source. To date, consistent results have
been obtained using HORM collagen (collagen reagent,
HORM, Hormon-Chemie, Germany; derived from
equine tendons, and containing predominantly Type 1
collagen), suspended at a final concentration of 50 ng/
mL in HORM buffer (isotonic glucose solution, pH 2.8)
containing 0.1 % sodium azide as a preservative. After the
collagen solution is added to wells (200 fiL per well),
plates are left undisturbed in a humidified container a
minimum of 48 hours (maximum 72 hours) at 22 °C to
obtain optimum coating. Plates are then washed, and
furthermore otherwise treated identically to that of the
VWF: Ag ELISA, excepting that TMB substrate color development was permitted to continue for 20 to 30 minutes. Test plasma values for VWF:CBA were calculated
with respect to PNP (= 1 U VWF/mL).
VWF Multimer Analysis
This was performed as previously described.1013'4
Briefly, electrophoresis of test plasma or serum was carried
out initially in a 2% agarose, discontinuous buffer/SDS system, and later in 1.5% and 3% gels to obtain additional
detail. Gels were run in a vertical apparatus (Hoefer, San
Francisco, CA), and after fixing were incubated with 125Ilabelled rabbit antibody to VWF (Dakopatts, Glostrup,
Denmark), washed thoroughly, dried onto Gelbond film
(FMC, Rockland, ME) and autoradiographed.
Fractionation of Plasma VWF
A glass chromatography column (BioRad, Australia;
2.5 cm X 42 cm) was coated with Cotasil (silicone coating material, Ajax Chemicals, Sydney, Australia) and
dried using a stream of nitrogen. Two hundred mL of
Sepharose 2B gel matrix (Pharmacia, Sydney, Australia)
was degassed under vacuum for 2 hours, and the matrix
then poured into the column in a single operation. The
column was equilibrated with 0.14 M NaCl, 0.02 M
HEPES (pH 7.0), and 10 mL of human plasma cryoprecipitate (enriched for VWF) was then applied to the top
of the column. A continuous flow of 0.14 M NaCl, 0.02
M HEPES (pH 7.0) was then applied to the column, and
the flow rate maintained at 0.5 mL per minute using a
peristaltic pump. Then 0.5 mL elute samples were collected and tested for both VWF:Ag and VWF:CBA (previously described) using a final dilution of 1:20.
Multimer analysis was also performed on selected eluate
samples. In some experiments, semiquantitation of
multimers was attempted. Following exposure to x-ray
film and autoradiography, individual lanes of an electrophoresed gel were sliced into 3 mm segments, and
counted using a gamma counter. The counts obtained
could then be related to the migration distance along the
gel as an estimate of multimer size and provided a basis
for the semiquantitation ofdifferent sized VWF forms.
RESULTS
Differences in VWF Levels in Plasma Versus Serum
Using either the VWF:Ag or VWF: RC of assays small
differences in assayed VWF could be detected in serum
samples compared to plasma samples derived from the
same individuals (Fig. 1A and Fig. IB, respectively).
However, using the VWF:CBA, a more dramatic decrease in the level of VWF was observed (Fig. 1C). Statistical comparison using paired analysis for nonparametric data (by the Wilcoxon signed rank test procedure)
gave the following P values when plasma derived VWF
A.J.C.P.- September 1995
FAVALORO, FACEY, AND GRISPO
Collagen Binding Assay for VWF
A. VWRAg
values were compared to serum derived VWF values:
VWF:Ag (P = .391); VWF:RCof (P = .019); VWF:CBA
(i><.001).
2.5
2.5
2
2-|
.1.
1.5
IT
i i i i i I i i i i I i i i i i i i i i i t i t i i t t
S
D
<
UJ
Although all three VWF assays were capable of detecting a decrease in VWF levels following nitration, the
VWF:CBA was most sensitive in this ability (Fig. 2), detecting large decreases in most samples assessed. Statistical comparisons using non-parametric paired analysis
(Wilcoxon signed rank test procedure) gave P values of
<.001 for all assay procedures.
1
%
0.5 >
0.5
0
Differences in VWF Levels in Plasma Following
Filtration
f 1.5
H I
1
U
M
Multimer Analysis of Serum and Filtered Plasma
B. VWF:RCof
To confirm that detected differences previously mentioned were because of removal of high molecular weight
forms in serum or following filtration, VWF:multimer
analysis was undertaken. Figure 3 shows sample comparisons with standard (ie, nonfiitered) plasma obtained
from individual donors and confirms reduction of (primarily high molecular weight forms of) VWF in serum
and filtered plasma samples obtained from the same donors.
2.5
2.5
2
2
1.5-1
1
•kS-
267
Hirp i.
0.5
1.5
1
-0.5
0
5 3
Assessment of VWF in Fractionated Plasma
Plasma VWF separated on the basis of molecular size
was detected differentially by the VWF:CBA assay, but
not using a standard VWF:Ag assay. As shown in Figure
4, VWF assessed in eluate fractions from a size exclusion
column was detected using a VWF:Ag assay in a manner
essentially unrelated to molecular size. In contrast, the
VWF:CBA detected VWF preferentially in early fraction
eluates. To confirm that this detection corresponded to
molecular size, multimer analysis undertaken on selected eluate fractions showed progressive loss of high
molecular weight forms of VWF with progressive elution
(see Fig. 5 for semiquantitatively depicted examples).
C. VWF:CBA
2.5
I M I
1.5
1.5
>
2.5
2
2-1
tu
•—•-
1
1
0.5
0.5-|
T — i—i"j-r
•0
DISCUSSION
S 3
•2 a:
< uj
FIG. 1. Differences in VWF values (y-axis, expressed in U/mL, with
respect to PNP [1 U VWF/mL]) comparing nonfiitered (ie, normally
processed) plasma samples versus serum samples obtained from the
same healthy normal donors for each assay studied (A: VWFrAg; B:
VWF:RCof; C: VWF:CBA). Left portion of eachfigureshows data as a
mean ± 1 SEM (n = 20; randomly selected normal donors); the right
portion of each figure shows individual sample data differences (• =
plasma, * = serum). To enable direct comparison between different
assay systems, VWF:CBA values (C) have been normalized to an upper
maximum value of 2.5 U/mL.
We have previously reported that refinements to the
VWF-collagen ELISA assay originally described by
Brown and Bosak,9 have permitted the use of this assay
(the "VWF:CBA") to consistently enable sensitive discrimination (far more so than the VWF:RCof assay) between plasma derived from Type 2 (subtypes 2A and 2B)
VWD patients, and that derived from Type 1 VWD patients, or from normal individuals. 10 " Type 2 VWD patients suffer a qualitative defect in VWF,5"8 which in
Vol. 104-No. 3
268
COAGULATION AND TRANSFUSION MEDICINE
Original Article
A. VWRAg
2.5
2.5
cr
2
2
E
=J1.5
B.
1.5$
1
K fai
T>'
ti:.,i
II1
> 0.5
0
~
E
T
i r r i i i i i i i i i i i i i 1 i~r i i i r i i l
<<
s s
<<
-J J
•
i. i. .
» •
•
1
uu
0.5 >
0
(NtNCNCNtN
a. a.
B. VWF:RCof
2.5
^
E
2.5
-2
2-|
~
E
1.5$
U,
A
1-1
> 0.5-1
0
1
1 **I tA
M *
\
a,
0.5 >
0
<<
<<
-1 _J
u- £
z
C. VWRCBA
2.5
CT
E
2-|
S 1-5-1
n,
•2
•
I
E
1.5^
•
1-
> 0.50-
2.5
"I
•
1
i
1
\
< <
T
I
1:
)
•
BU
0.5 >
0
-nNMNM
< <
-J
J
a. a.
it it
z
FIG. 2. Differences in VWF values (y-axis, expressed in U/raL, with
respect to PNP [1 U VWF/mL]) comparing nonfiltered (ie, normally
processed) plasma samples versus identical plasmas following filtration
through standard 0.22 n filters for each assay studied (A: VWF:Ag; B:
VWF:RCof; C: VWF:CBA). Left portion of figure shows data as mean
± 1 SEM (nonfiltered plasma: "NF/PLASMA;" filtered plasma: " F /
PLASMA;" n = 24; randomly selected normal donors); the right portion of each figure shows individual sample data differences ( • =
plasma, * = filtered plasma). To enable direct comparison between
different assay systems, VWF:CBA values (C) have been normalized to
an upper maximum value of 2.5 U/mL.
Type 2A (and to a lesser extent in Type 2B) VWD patients is associated with an absence of high molecular
weight forms of VWF in plasma (ie, those forms most
functionally relevant or active). In contrast, Type 1
VWD patients suffer a partial quantitative deficiency of
VWF, with all molecular weight forms essentially present, but in a reduced level compared to normal individuals.5"8 Clinically, discrimination between the two types
is important as therapeutic management of such patients
will differ.7'8 That the VWF:CBA can be used to discriminate between these VWD subtypes is therefore an important laboratory advancement. Although it had been
proposed by us that the reason for this behavior lay in
the selective basis by which the VWF:CBA could detect
high molecular weight forms of VWF, this had never
been formally assessed. This report, in part, deals with
this by providing direct evidence for preferential recognition of the VWF:CBA for these high molecular weight
forms of VWF (Figs. 4 and 5).
The current study has also evaluated these three laboratory VWF assays in terms of their ability to recognize
VWF in serum or filtered plasma compared to standard
(nonfiltered) plasma. We found that the VWF:CBA was
most sensitive among the assays in its ability to consistently detect a significant decrease in VWF levels in serum compared to plasma (Fig. 1). If the dilutional effect
of plasma is taken into account (ie, 9 parts whole blood
collected into 1 part citrate), then the VWF:Ag and the
VWF:RCof assays were certainly capable of detecting
small falls in the VWF level in serum. However, such
detected differences are completely overshadowed by
that observed using the VWF:CBA. In normal samples,
serum VWF:Ag levels have previously been reported in
some studies to be quantitatively the same as that observed in plasma.1516 These reports used low sensitivity
VWF assays, and VWF is known to cross-link to fibrin
during clot-gel formation 17. Furthermore, binding of
VWF to cross-linked or noncross-linked fibrin is known
to be dependent on VWF multimer size,18 and VWF can
facilitate platelet incorporation into polymerizing fibrin
independent of ristocetin.19 Taken together, and in view
of our previous data on selective discrimination between
VWD subtypes,10" and other evidence indicating that
high molecular weight forms of VWF preferentially bind
to collagen (this report and references 19-21), we suggest
that the reason that the VWF:CBA can so clearly detect
differences in VWF levels between serum and plasma,
compared to the VWF:Ag and VWF:RCof assays, is that
the VWF:CBA selectively recognizes VWF of higher molecular weight forms. In support of this view, comparative multimeric analysis performed on derived individ-
A.J.C.P. • September 1995
FAVALORO, FACEY, AND GRISPO
Collagen Binding Assay for VWF
1 2
3
4
1 2
**
3
4
5
269
6
7
8
<*&• ,m. ••:
A.
B.
FlG. 3. Sample VWF:multimer analysis for material derived from 6 separate individual normal donors ("IND"). Multimers run from high molecular weight forms (top offigure)to low molecular weight forms (bottom offigure).A: Lanes 1 and 2: IND'M" as serum (Lane l)and plasma (Lane
2); Lanes 3 and 4: IND "2" as serum (Lane 3) and plasma (Lane 4); B: Lanes 1 and 2: IND "3" as serum (Lane 1) and plasma (Lane 2); Lanes 3 and
4: IND "4" as serum (Lane 3) and plasma (Lane 4); Lanes 5 and 6: IND "5" asfilteredplasma (Lane 5) and nonfiltered plasma (Lane 6); Lanes 7
and 8: IND "6" asfilteredplasma (Lane 7) and nonfiltered plasma (Lane 8).
ual plasma and serum does indicate removal of higher
molecular weight forms in the latter (Fig. 3). This finding
is significant because these forms of VWF are known to
be most functionally relevant (eg, higher molecular
weight multimer forms are the most active in supporting
platelet aggregation22,23 and adherence,24 and also in the
ability to bind to elements of the vascular cell wall in-
E
3
<
LL
>
" O O O O O O O O O O O O O O O O O O C K
T-c\ico-*irtcoi^cococ>T-c\ico'<*m<or"-co
Fraction number
FIG. 4. Levels of VWF determined in column eluates. Figure shows
fraction number (x axis), and VWF levels as assessed by the VWF:CBA
assay (left y axis) and the VWF:Ag assay (right y axis) given as U/mL
equivalent of undiluted fraction material (with respect to PNP providing 1 U VWF/mL equivalent).
eluding collagen,19"21 sulphated glycolipids,25 and extracellular matrix components26).
Furthermore, our finding that VWF levels decreased
in plasma following filtration through a standard 0.22 ft
filter (Fig. 2) is also an important laboratory finding.
Again, although some evidence of sensitivity to detect a
change in VWF levels was present using both the VWF:
Ag and VWF:RCof assays, the VWF:CBA was most sensitive in its ability to detect the change. Importantly,
VWF levels detected in filtered plasma using the VWF:
Ag and VWF:RCof assays tended to remain within the
normal reference ranges for these assays (0.5-2.0 U/mL;
see references 10, 11, whereas those detected using the
VWF:CBA tended to decrease below the normal reference range for this assay (0.5-2.5 U/mL). 10 " von Will'ebrand factor is an adhesive protein, and during the filtration process is probably adhering onto thefiltermatrix
(rather than physically excluded on the basis of size).
Again, the likely explanation is that high molecular
weight forms of VWF are more readily trapped by the
filtration process, because these are the most adhesive
forms. In support of this view, comparative multimeric
analysis performed on individual plasma samples preand post-filtration does indicate removal of higher molecular weight forms in the latter (Fig. 3).
These findings highlight important ramifications for
laboratories performing diagnostic VWF/VWD studies.
There will be occasions where filtered plasma (or possi-
Vol. 104-No. 3
270
COAGULATION AND TRANSFUSION MEDICINE
Original Article
bly serum) is provided to the laboratory for VWF evaluation (and potentially unknowingly received in that
manner into the laboratory). A typical working example
is the follow-up investigation of an otherwise unexplained abnormal APTT (activated partial thromboplastin time, or equivalent "screening" contact mechanism
pathway clotting test) obtained during routine laboratory coagulation screening. Subsequent combined testing of such samples for lupus anticoagulants and anticardiolipin antibody status together with VWF workups
may be requested to help explain the prolonged APTT
and exclude "lupus-like" disorders and VWD, respectively.27 Unfortunately, residual platelet material will interfere with lupus anticoagulant assays, and consequently the recommendation from the relevant
international committee for the standardization of lupus
anticoagulants28 recommends "nitration through 0.22 n
screens to remove platelets prior to freezing" and subsequent screening. Similarly, serum is the recommended
specimen for anticardiolipin antibody assays, and in
•*• Fraction 10
1,200
•+• Fraction 30
• Fraction 40
c 1,000
Counts
Q.
800
600
400
200
0
O
^3
<
03
5> 22
1 . 500
< 1li.'
„ 0^
V
«V
.^
<&
^
oV
^&
^
oV
FIG. 6. Data obtained for VWFrAg to VWF:CBA ratio (ie, ratio of
VWF:Ag derived values to VWF:CBA derived values) expressed as
mean ± 1 SEM for samples identified in Figures 1 and 2. On an appropriate nonfiltered plasma specimen, a VWF:Ag to VWF:CBA ratio of
>2.5 can generally be concluded as deriving from a patient with Type
2A or Type 2B VWD (reference 10, 11). Similarly, however, an inappropriately assayed serum or filtered plasma derived from a normal
individual could also be concluded as having derived from such an individual, with potential consequent misdiagnosis.
-•-Fraction 120 * Fraction 180
'E
CD
4.5
10 20 30 40 50 60 70 80 90 100
Migration Distance (mm)
FIG. 5. Semiquantitation of VWF:multimers present in sample eluate
fractions depicted in Figure 4. Following exposure to x-ray film and
autoradiography (data not shown) individual lanes of an electrophoresed gel were sliced into 3 mm segments, and counted using a gamma
counter. Thefigureshows averaged counts obtained (counts per minute, y axis), in relation to migration distance along the gel (mm; x axis),
and representing the range of high molecular weight VWF forms (at
low migration distance) to intermediate molecular weight VWF forms
(at intermediate migration distance) to low molecular weight forms (at
high migration distance). As can be seen, early fractions (eg, fractions
30 and 40) have correspondingly greater amounts of high molecular
weight forms and less of low molecular weight forms; later fractions (eg,
fractions 120 and 180) have greater amounts of lower molecular weight
forms, and less of high molecular weight forms. This pattern corresponds to both x-ray autoradiography (not shown), and to the differential pattern observed using the VWF:CBA and VWF:Ag assays
(Fig. 4).
many institutions including ours, this testing is performed in the same laboratory as those of lupus and
VWF testing. Should VWF studies be performed on such
specimens (serum orfilteredplasma), a false diagnosis of
VWD may arise either from the laboratory which (unknowingly) performed these studies, or from the referring clinician attempting to interpret the laboratory
findings. This may arise particularly if communication
between the testing laboratory and the referring site is
poor, if the specimen collection and processing site is
separate or distant to the laboratory testing site, or if conclusive patient clinical history is absent, and a VWFrAg
to VWF:CBA ratio is determined and found to be inappropriately high. Previous studies have shown that a
VWF:Agto VWF:CBA ratio of >2.5 is otherwise suggestive of Type 2A or Type 2B VWD. 10 " Such VWF:Ag to
VWF:CBA ratios can be obtained with normal individuals if serum or filtered plasma is inadvertently tested
(Fig. 6).
In conclusion, the current report details recent studies
to further highlight the unique and important properties
of the VWF:CBA, and suggests caution during the interpretation of abnormal results in the absence of information regarding the true clinical history of the patient under evaluation, or in the absence of information
regarding the true nature of the "plasma" specimen re-
A.J.C.P.-September 1995
FAVALORO, FACEY, AND GRISPO
Collagen Binding Assay for VWF
ceived and tested. Indeed, it should be noted that filtered
plasma and serum are not appropriate specimen types
for VWF studies, and any abnormal finding should be
considered as potential artifacts of such testing.
Acknowledgments.
The authors thank Tammie Browning and
Trudi Posa for "number crunching," and Judith Maybin for expert
typographical assistance in the original preparation of the manuscript.
The authors also thank the staff of the Parramatta Red Cross Blood
Bank, without whose help such work would not be possible; and Dr.
Thomas Exner for access to some of the multimer analyses performed
by him.
REFERENCES
1. Moroose R, Hoyer LW. von Willebrand factor and platelet function. Ann Rev Med 1986;37:157-163.
2. Giddings JC, Peake IR. The investigation of factor VIII deficiency.
In: Thompson JM, ed. Blood Coagulation and Haemostasis.
New York: Churchill Livingstone, 1985, pp 135-207.
3. Girma J-P, Meyer D, Verweij CL, et al. Structure-function relationship of human von Willebrand factor. Blood 1987;70:605611.
4. Sadler JE. Review: von Willebrand factor. J Biol Chem 1991; 266:
22777-22780.
5. Sadler JE, Gralnick HR. Commentary: A new classification for
von Willebrand disease. Blood 1994;84:676-679.
6. Sadler JE. A revised classification of von Willebrand disease.
ThrombHaem 1994;71:520-525.
7. Mannucci PM, Altieri D, Faioni E. Vasopressin analogues: Their
role in disorders of haemostasis. In: Lusher JM, ed. Annals of
the New York Academy of Sciences, vol 509, 1987, pp 71 - 8 1 .
8. Rodeghiero F, Castamann G, Mannucci PM. Clinical indications
for desmopressin (DDAVP) in congenital and acquired von
Willebrand disease. Blood Reviews 1991;5:155-161.
9. Brown JE, Bosak JO. An ELISA test for the binding of von Willebrand antigen to collagen. Throm Res 1986;43:303-311.
10. Favaloro EJ, Grispo L, Exner T, Koutts J. Development of a simple collagen-based ELISA assay aids in the diagnosis of, and permits sensitive discrimination between Type I and Type II, von
Willebrand's disease. Blood Coagul Fibrinolysis 1991;2:285291.
11. Favaloro EJ, Grispo L, Dinale A, et al. von Willebrand's Disease:
Laboratory investigation using an improved functional assay for
von Willebrand factor. Pathology 1993;25:152-158.
12. Favaloro EJ, Dean M, Grispo L, Exner T, Koutts J. von Willebrand's Disease: Use of collagen binding assay provides potential improvement to laboratory monitoring of Desmopressin
(DDAVP) therapy. AmJHematol 1994;45:205-211.
13. Exner T, Hill P, Cleland J, et al. Studies on an unusual von Willebrand's variant-type UD.Aust NZJMed 1990;20:553-557.
271
14. Ruggeri ZM, Zimmerman TS. Variant von Willebrand's disease:
Characterization of two subtypes by analysis of multimer composition of factor VHI/von Willebrand factor in plasma and
platelets. J Clin Invest 1980;65:1318-1325.
15. Hada M, Kato M, Ikematsu S, et al. Possible cross-linking of factor
VIII related antigen to fibrin by Factor XIII in delayed coagulation process. ThrombRes 1982;25:163-168.
16. Giddings JC, Hogg S, Legg IR, Hughes IA. The relationship between von Willebrand Factor antigen and fibronectin in human
plasma, endothelial cells, and fibroblasts in culture. Thromb Res
1986;44;291-301.
17. Hada M, Kaminski M, Bockenstedt P, McDonagh J. Covalent
crosslinking of von Willebrand factor to fibrin. Blood 1986;68:
95-101.
18. Ribes JA. Francis CW. Multimer size dependence of von Willebrand factor binding to cross linked or non-crosslinked fibrin.
Blood 1990;75:1460-1465.
19. Kassler CM, Floyd CM, Rick ME, et al. Collagen-factor VHI/von
Willebrand factor protein interaction. Blood 1984; 63:12911298.
20. Scott CM, Griffin B, Pepper DS, et al. The binding of purified factor VIII von Willebrand factor to collagens of differing type and
form. Throm Res 1981;24:467-472.
21. Santoto SA. Preferential binding of high molecular weight forms
of von Willebrand factor to fibrillar collagen. Biochem Biophvs
Acta 1983;756:123-126.
22. Doucet de Bruine MHM, Sixma JJ, Over J, Beeser-Visser NH.
Heterogeneity of human factor VIII. II. Characterization of
forms of factor VIII binding to platelets in the presence of ristocetin. J Lab Clin Med 1978;92:96-107.
23. Moake JL, Turner NA, Stathopoulos NA, et al. Involvement of
large plasma von Willebrand factor (VWF) multimers and unusually large VWF forms derived from endothelial cells in shear
stress induced platelet aggregation. J Clin Invest 1986; 78:14561461.
24. Sixma JJ, Sakariassen KS, Beeser-Visser NH, et al. Adhesion of
platelets to human artery subendothelium: Effect of Factor VIIIvon Willebrand factor of various multimeric composition.
Blood 1984; 63:128-138.
25. Roberts DD, Williams SB, Gralnick HR, Ginsburg V. von Willebrand factor binds specifically to sulfated glycolipids. J Biol
Chem 1986;261:3306-3309.
26. Sporn LA, Marder VJ, Wagner DD. von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively. Blood 1987;69:
1531-1534.
27. Favaloro EJ. Assessment of haemostatic function: Follow-up evaluation of abnormal screening coagulation tests and possible outcomes. Aust J MedSci 1994; 15:39-45.
28. Exner T, Triplett DA, Taberner D, Machin SJ. Scientific and standardisation committee communication: Guidelines for testing
and revised criteria for lupus anticoagulants. SSC subcommittee
for the standardization of lupus anticoagulants. Thromb Haem
1991;65:320-322.
Vol. 104-No. 3