Coagulation and Transfusion Medicine / DETECTION OF QUALITATIVE VON WILLEBRAND DISORDER SUBTYPES
Detection of von Willebrand Disorder and Identification of
Qualitative von Willebrand Factor Defects
Direct Comparison of Commercial ELISA-Based von Willebrand
Factor Activity Options
Emmanuel J. Favaloro, PhD
Key Words: von Willebrand factor; vWF; von Willebrand disease; von Willebrand disorder; vWD; Diagnosis; Collagen binding assay;
Collagen; vWF:CBA; Qualitative defects
Abstract
Two von Willebrand factor (vWF):collagen binding
(activity) assay (CBA) kit methods are commercially
available. A monoclonal antibody (MAB)based
enzyme-linked immunosorbent assay (ELISA) system
reported to correlate with a standard vWF:ristocetin
cofactor (RCof) assay is also commercially available. It
is marketed as a vWF:Activity assay and is available in
2 assay version formats. In the present study, these 4
vWF-activity options were compared directly with in-
house vWF:CBA ELISAs for their ability to detect von
Willebrand disease (vWD) and identify qualitative vWF
defects. The 2 MAB-based systems detected vWD but
could not specifically identify qualitative vWF defects,
although the recently modified Mark II kit was more
effective for the latter compared with the original Mark
I kit. All vWF:CBA methods, including in-house and
commercial, also effectively detected vWD but differed
in their ability to identify qualitative vWF defects.
Effectiveness was highest using the in-house reference
vWF:CBA (using a type I/III collagen mix product from
equine tendon), the Gradipore vWF:CBA (also uses
equine tendon-derived collagen), or the in-house
vWF:CBA methods using type III human collagen at a
relatively low concentration (1 or 3 g/mL, without
covalent linkage). The IMMUNO vWF:CBA seemed to
be the least effective among the vWF:CBA methods for
detection of qualitative vWF defects.
608 Am J Clin Pathol 2000;114:608-618
von Willebrand disorder (von Willebrand disease,
vWD), is the most common inherited bleeding ailment.1-3
People with vWD have defects in or reduced levels of von
Willebrand factor (vWF), an adhesive plasma protein
required for effective primary hemostasis. 4 vWD is a
heterogeneous disorder, and patients are subtyped according
to pathophysiology, using clinical and laboratory criteria.2,3
Briefly, type I vWD is a partial quantitative deficiency in
vWF, type III vWD a total quantitative deficiency in vWF,
and type II vWD a qualitative abnormality or deficiency in
vWF. Specific identification of qualitative vWF defects (ie,
type II vWD) is considered important because of differen-
tial disease management.2,3,5-10
Although types I and III vWD usually can be detected
using a vWF:antigen (Ag) assay, patients with type II vWD
can have normal plasma vWF:Ag levels. Accordingly,
specific identification of type II vWD requires additional
testing, inclusive of a functional vWF (activity) assay. The
typical laboratory pattern for plasma from type II vWD
subtypes is termed vWF-discordant. Thus, for type IIA, IIB,
and IIM, vWF levels determined using the functional/activity
vWF assay are typically much lower than those determined
using the vWF:Ag. Collectively then, it should be possible to
detect all forms of vWD and specifically identify a qualitative
vWD subtype, using a diagnostic screening process that uses
a standard (quantitative, nonfunctional) vWF:Ag assay part-
nered with an effective quantitative functional vWF assay and
a factor VIII assay. Classically, the vWF:ristocetin cofactor
(RCof) assay represents the original functional/activity vWF
assay. Alternative functional/activity vWF assays have more
recently been described and are available commercially.8-26
The vWF:collagen-binding activity (CBA) assay is one
option. In the authors laboratory, the vWF:CBA consistently
American Society of Clinical Pathologists

has proved superior to the vWF:RCof assay for the identifi-
cation of type II vWD,8,11-14 has reduced assay variability
(interassay and interlaboratory),8,11,15 and is a better marker
of desmopressin responsiveness.16 Some of these findings
have been confirmed by others. 17-21 Enzyme-linked
immunosorbent assay (ELISA)based vWF:CBA kits are
available commercially from 2 manufacturers (Gradipore,
Sydney, Australia, and IMMUNO, Heidelberg, Germany),
but independent evaluations of their usefulness are lacking.
This is unfortunate, since laboratories often choose a
commercial option in preference to an in-house procedure
and since the type and concentration of collagen used in such
an assay are of critical importance in the efficacy of the assay
for identifying qualitative vWD subtypes.22
Another potential alternative to vWF:RCof (by platelet
agglutination) also has been described.23-25 Based on a
monoclonal antibody (MAB) vWF capture system, the assay
forms the basis of a popular commercial kit method (Shield
Diagnostics, Dundee, Scotland). In developmental studies
and using plasma from patients with type I and II vWD,
correlation with vWF:RCof (agglutination) was better than
that seen with vWF:Ag.23-25 Because of this, and because the
MAB used in the assay inhibits ristocetin-induced platelet
aggregation and reportedly binds vWF at the platelet Gp-Ib
binding site (ie, is a vWF function inhibitor), the commercial
kit is marketed as a vWF:activity ELISA.
Unfortunately, the promising developmental studies23-25
have not been confirmed by a number of independent
studies using the commercial kit.11,19,22,26 The reason for
this inconsistency has been explained only recently. The
authors laboratory evaluated the Shield Diagnostics
commercial kit in parallel with in-housedeveloped MAB
anti-vWF assay systems using locally generated MABs.27
The MAB systems identified qualitative vWD subtypes, but
specific identification was highly dependent on the way
that the assay was set up. Thus, some in-house MAB-based
systems were able to specifically identify qualitative vWD
subtypes, but the commercial assay essentially failed to do
so. 27 Concurrently, following other user feedback, the
manufacturer has modified the assay by altering the detec-
tion system. The manufacturer reports that, compared with
the previous assay, the modified version correlates better
with vWF:RCof.28 However, independent published evalua-
tions of this modified assay system are lacking, and the
modified kit was unavailable to the authors laboratory at
the time of previous evaluations.27
Accordingly, each of the commercially available
ELISA-based vWF-activity options were compared directly
for their effectiveness in detecting vWD and specific qualita-
tive vWF defects. The usefulness of the 2 separate commer-
cially available vWF:CBA kit methods and the commercially
available MAB-based kit assays (ie, Shield vWF:Activity,
American Society of Clinical Pathologists
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
both assay versions) have been compared directly, together
with various in-house vWF:CBA assay methods. There is
notable variability in the effectiveness of different procedures
to detect qualitative vWD defects (ie, type II vWD).
Materials and Methods
Sample Testing
All vWD samples were derived from patients diagnosed
as having vWD using standard criteria2,3 and obtained with
informed consent. All vWD samples were assessed for factor
VIII, vWF:Ag, vWF:CBA, and vWF:RCof.8,12,13,22 Further
subtyping analysis was undertaken whenever possible in
consultation with the referring clinician and using
vWF:multimer analysis and/or ristocetin-induced platelet
aggregation testing as appropriate.8,12,13,22 In some cases,
patients were further assessed using DNA analysis.29,30
Evidence of vWF discordance (ie, qualitative or functional
vWF defect) was noted whenever relevant.8,12,13,22 In addi-
tion to vWD plasma, a number of normal samples were
collected and used for comparative studies, as well as for
generation of a pooled normal plasma (typically >60
persons). All individual plasma samples were prepared
following collection into standard buffered sodium citrate
tubes (0.105-mol/L concentration of citrate: citrate/blood,
1:9) and centrifuged (1,200g; 15 minutes) to isolate plasma.
Samples were frozen in aliquots at 80C until required for
comparative studies.
Laboratory Studies
Methods for standard assay procedures (eg, vWF:Ag,
vWF:CBA, vWF:RCof) have been published. 8,12,13,22
vWF:RCof was performed using a standard agglutination
assay with fixed platelets, whereas vWF:Ag and vWF:CBA
were performed using ELISA.8,12,13,22 Commercial ELISA kit
procedures comprised similar standard sandwich ELISA
techniques. All manufacturers agreed to provide 2 or more
kits from a single current lot of their product for evaluation.
The present study coevaluated the following commercial
options, all of which were used according to manufacturers
instructions:
1. vWF:activity ELISA (functional anti-vWF MAB-based;
Shield Diagnostics; supplied by Dade-Behring, Australia);
the current version 1 (Mark II, the newly modified procedure,
product code FvWF200) and the current version 2 (Mark I,
the original procedure, product code FvWF100) kits were
tested. Both are similar but differ in terms of the assay detec-
tion system. In the Mark I kit, a horseradish peroxidase
(HRP)conjugated rabbit anti-vWF is used; in the Mark II kit,
HRP-conjugated MAB anti-vWF is used.
Am J Clin Pathol 2000;114:608-618 609
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2. vWF:CBA test kits from (a) IMMUNO AG
(IMMUNOZYM vWF:CBA, distributed by PROGEN
IMMUNO-Diagnostika, Heidelberg, Germany) and (b)
Gradipore (Collagen Binding Assay, product code CBAE-1,
Gradipore, Sydney, Australia).
The sandwich ELISA process initially involves coating
plates with material capable of binding vWF (ie, collagen or
antibody = first layer, which enables vWF capture). After
incubation and washing, plasma (containing vWF) is added,
and vWF subsequently is bound to plates (the second layer,
ie, vWF captured by first layer). After further incubation and
washing, a third layer is introduced, this time comprising the
antibody to vWF, and together with subsequent additional
layers forming the vWF detection system. Thus, in the
vWF:Ag procedure, plates are coated with rabbit anti-vWF
antisera (R-anti-vWF; DAKO, Sydney, Australia), followed
by sequential washing and incubation steps using plasma
(source of vWF), HRP-labeled R-anti-vWF (DAKO), and
color generation using tetramethyl benzidine hydrochloride
as HRP-substrate. 8,12,13,22 The in-house vWF:CBA, the
commercial vWF:CBA assays, and the commercial MAB-
based kit methods are technically similar but respectively use
collagen or an MAB against vWF as the first layer/vWF-
capture process instead of R-anti-vWF. This is summarized
in Table 1.
Unless otherwise stated, conditions for all in-house
ELISAs (eg, sample and reagent incubation times, blocking
buffer, washing steps, washing buffer, plasma dilutions,
HRP-substrate, and ELISA plates) were essentially identical
and comparable to those used in standard vWF:Ag and
vWF:CBA assays at the authors laboratory.8,12,13,22 For the
vWF:Ag assay, rabbit antibody to vWF (Dakopatts, Sydney,
Australia) is diluted in a 0.1-mol/L concentration of
NaHCO3 buffer (pH 8.3) for coating ELISA plates (EIA
plates, ICN, Sydney, Australia; 200 L per well; plate left
covered overnight at 4C in a wet-box before use on the day
of assay). For vWF:CBA, the reference method involved
using Nycomed-HORM collagen (Nycomed Arzneimittel,
Ismaning, Germany; 1 mg collagen per milliliter of stock)
diluted to a final concentration of 50 g/mL using HORM
buffer containing 0.1% sodium azide and left to coat onto
ELISA plates (200 L of collagen solution per well) for 4
days at room temperature in a wet-box. The HORM collagen
source is derived from equine tendon and is described by the
manufacturer as a type I/type III collagen mixture (95%/5%,
respectively). As an alternative to the Westmead Hospital
Laboratory reference vWF:CBA method, type III collagen
(derived from human placenta; Sigma, Sydney, Australia;
code number C4407; lot number 65H39041) also was used
for comparative studies as previously described22 (NB: this
type III collagen is believed to be similar to that used in the
IMMUNO CBA kit).
On the day of use, all in-housederived assay plates
were washed 3 times with wash buffer and blocked for 1
hour using 5% bovine serum albumin. Thereafter, plates
were treated according to a standard vWF ELISA procedure
(see elsewhere for additional details).8,12,13,22 Unless other-
wise stated, patient plasma samples were tested in triplicate
for each experiment at a final dilution of 1:100. Pooled
normal plasma (deemed to contain 100% vWF) was used to
generate comparative calibration curves for each in-house
assay. In addition, control plasma samples (in-house
[including cryosupernatant] and commercial [eg, assayed
reference plasma, SARP, Helena, Mulgrave, Australia]) were
used in each experiment.
All commercial kits were used according to the manu-
facturers instructions. To more fairly compare test results
from all assays, however, and as standardly performed for
the Westmead Hospital Laboratory in-house method,
samples were tested in triplicate rather than in duplicate as
suggested in the commercial product inserts. Calibration
curves were generated using kit-provided material but

Table 1
Summary of Enzyme-Linked Immunosorbent Assay (ELISA) Steps for Each Coevaluated Procedure

Layer*

Assay Type 1 (vWF Capture) 3 (vWF Detection)

vWF:Ag R-anti-vWF HRP-R-anti-vWF

vWF:CBA (in-house reference) Equine collagen (type I/III mix) HRP-R-anti-vWF
vWF:CBA (in-house alternative type III collagen) Human type III collagen (placental derived) HRP-R-anti-vWF
vWF:CBA (IMMUNO) Human type III collagen HRP-R-anti-vWF
vWF:CBA (Gradipore) Equine collagen (type not specified) HRP-R-anti-vWF
vWF:activity (version 1 [Mark II]) MAB-anti-vWF HRP- MAB-anti-vWF
vWF:activity (version 2 [Mark I]) MAB-anti-vWF HRP-R-anti-vWF

CBA, collagen-binding (activity) assay (type III collagen was evaluated using 3 collagen-coating concentrations: 1, 3, and 50 g/mL; HRP-MAB-anti-vWF, horseradish
peroxidaselabeled MAB-anti-vWF; HRP-R-anti-vWF, horseradish peroxidaselabeled R-anti-vWF; MAB, monoclonal antibody; MAB-anti-vWF, MAB against vWF; R-anti-
vWF, rabbit antisera to vWF. Mark I and Mark II are Shield Diagnostics ELISA kits (Dundee, Scotland); IMMUNO, Heidelberg, Germany; Gradipore, Sydney, Australia.
* Layer 2 is identical for each assay (ie, plasma as a source of vWF).

610 Am J Clin Pathol 2000;114:608-618 American Society of Clinical Pathologists


normalized if required to the percentage of vWF (ie, where 1
U/mL is defined as equivalent to 100% vWF). Kit-provided
controls also were run with each assay, in addition to other
controls (ie, pooled normal plasma, SARP, cryosupernatant).
Data derived for all tested controls for all assays were consis-
tently within stated or expected limits.
In general, multiple assays were performed on the same
or sequential days using freshly thawed or reconstituted
samples, and most tested samples were tested in all assays
(ie, same sample sets used for most assays). In addition,
some samples were retested in the same assay type on
different days to help assess reproducibility. However,
because of variations to the recommended construction of
calibration curves for the commercial kits and the recom-
mended use of commercial controls, additional incorporation
of other controls, and limited availability of the commercial
kits, not all samples were tested in all assays. Accordingly, as
indicated in the Results section, sample numbers tested
between assays on occasion varied slightly. This is not
expected to unduly influence interpretation of results.
Statistical Analysis
All data were analyzed using GraphPad Prism 2.0C
(GraphPad Software Inc, San Diego, CA) for the Macintosh.
Data obtained for samples retested in the present study also
were sometimes compared with historic data obtained on
samples before storage (ie, to identify potential variance).
Results
Detection of vWF by Various Assays
All assays, both commercial and in-house, were
capable of detecting plasma vWF in a similar vWF dose-
dependent manner (ie, serial dilutions of plasma standard
generated good and comparable vWF concentration-depen-
dent calibration curves for all assays as characterized by
relatively fast substrate color development, high ELISA
optical density readings, and steep upward linearity with
respect to the vWF-concentration at the clinically relevant
vWF range Figure 1). There was also generally excellent
reproducibility for each calibration curve for each method.
End-stage color generation was also acceptable for each
assay, based on the tetramethyl benzidine hydrochloride
substrate incubation/color generation time (between 5 and
30 minutes for each assay) and the final optical density
reading.
Detection of vWD by Various Assays
All assays, both commercial and in-house, were gener-
ally capable of detecting vWD (ie, plasma samples from
American Society of Clinical Pathologists
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
patients with identified vWD generally gave assay values
that were below standard reference ranges, although some
difference between assays was noted, particularly for type
II vWD Figure 2A).
Correlation Analysis
This was undertaken as a means to compare procedural
endpoints for similarities (ie, do derived test results for
different vWF assays compare well overall?). Summary
data are given in Table 2. As can be seen, there was good
correlation between most assays. For example, combined
sample test data for vWF:Ag correlated well with all other
assays, although the greatest correlation was with historic
vWF:Ag data (ie, correlation with alternative vWF assays
was good, but notably weaker). Similarly, combined sample
test data for the in-house reference vWF:CBA correlated
well with all other assays, although correlation was best for
the historic in-house vWF:CBA data, Gradipore vWF:CBA
data, and in-house vWF:CBA assays using type III collagen
(ie, correlation with alternative vWF assays was good, but
notably weaker).
Although good correlation between vWF assays also was
observed when data were restricted to specific patient groups,
some additional differences were noted. For example, for the
type II vWD sample group, correlation of vWF:Ag data with
historic vWF:Ag was good but somewhat less strong
compared with functional vWF test data such as vWF:CBA.
Similarly, for the type II vWD sample group, correlation of
in-house vWF:CBA data with historic in-house vWF:CBA
and other functional vWF assays was good but was somewhat
less strong compared with vWF:Ag test data.
Specific Identification of Qualitative vWF Defects
Although correlation analysis provides a means to test
for similarity of data, it will not identify whether different
vWF test systems are capable of specifically identifying
qualitative vWF defects. The authors laboratory previously
has shown the value of determining the ratio of vWF:Ag to
vWF:CBA to help distinguish type I from type II vWD, as
high ratios (ie, >2.0) generally are consistent with type II
vWD and essentially reflect discordance in derived vWF
data between functional and nonfunctional vWF for people
with type II vWD.8,9,11-13,22 Accordingly, to assess the rela-
tive strength of each functional/activity vWF assay to differ-
entially identify a qualitative vWF disorder, results obtained
using plasma from patients with type I or type II (IIA or IIB
phenotypes) vWD were compared in terms of their vWF:Ag
to vWF:other assay ratios for all assay systems evaluated in
this study Figure 2B. As shown, different assay systems
differed in their relative power to specifically identify type II
vWD as distinct from type I vWD, although group data for
all functional/activity vWF assays showed a differential
Am J Clin Pathol 2000;114:608-618 611
Favaloro / DETECTION OF QUALITATIVE VON WILLEBRAND DISORDER SUBTYPES

A B

1.4 2.2

2.0
1.2
1.8

1.6
1.0

1.4

0.8

OD
1.2
OD

1.0
0.6

0.8

0.4
0.6

0.4
0.2
0.2

0.0 0.0

0 25 50 75 100 125 150 175 200 0 25 50 75 100 125 150 175 200

VWF (%) VWF (%)

C Figure 1 von Willebrand factor (vWF)-dose dependent

calibration curves generated using various assay procedures
and serial dilutions of calibration plasma (x-axis, range, 0%-
200% vWF, where pooled normal plasma [representing the
1.4 equivalent of 100% of normal] was used for in-house
procedures, and a kit provided calibration plasma samples
1.2 used for commercial kits). All assay results normalized to
percentage of vWF (where 1 U/mL = 100% vWF). Each point
1.0 reflects the mean of triplicate values; each line represents data
from a separate experimental enzyme-linked immunosorbent
assay (ELISA) plate. A, vWF:antigen (Ag) and in-house (IH;
0.8
reference method) vWF:collagen-binding (activity) assay (CBA).
OD

B, Commercial vWF:CBA methods (IMMUNO [IM],

0.6
Heidelberg, Germany, and Gradipore [Grad], Sydney, Australia).
C, Shield Diagnostics ELISA (SE; Dundee, Scotland) assays
0.4 (version 1 [V1], Mark II; version 2 [V2], Mark I). D, In-house
vWF:CBA using an alternative collagen source (ie, human type
0.2 III collagen) at 3 collagen-coating concentrations (ie, 1, 3, and
50 g/mL). OD, optical density.
0.0
0 25 50 75 100 125 150 175 200

VWF (%)

pattern. That is, best separation of type II vWD data from
type I vWD was obtained using the reference in-house
vWF:CBA method (historic data and data from the present
study yielded similar findings). Good separation generally
also was obtained using the Gradipore vWF:CBA and the
in-house human type III collagen vWF:CBA assays when
the collagen-coating concentration was used at a relatively
low level (ie, data for 1 and 3 g/mL yielded better separa-
tion of vWD subgroups compared with data for 50
g/mL). Although group data for the IMMUNO
vWF:CBA assay and the Shield vWF:Activity assays
showed a trend toward separation, this was not as complete

612 Am J Clin Pathol 2000;114:608-618 American Society of Clinical Pathologists

 Coagulation and Transfusion Medicine / ORIGINAL ARTICLE

A 80

70

60

50
VWF (%)

40

30

20

10

0
Historical
VWF:Ag

Historical
VWF:CBA

Historical
VWF:RCof

Current Study
VWF:Ag

Current Study
VWF:CBA (IH)

Gradipore
VWF:CBA

IMMUNO
VWF:CBA

IH-VWF:CBA
III-1 g/mL

IH-VWF:CBA
III-3 g/mL

IH-VWF:CBA
III-50 g/mL

Shield-V1
"VWF:Activity"

Shield-V2
"VWF:Activity"
Assay/Collagen Type

Type 1 VWD Type 2 VWD

B 10

6
VWF (%)

0
Historical
Ag/CBA

Historical
Ag/RCof

Current Study
Ag/IH-CBA

Ag/Graqdipore
CBA

Ag/IMMUNO
CBA

Ag/IH-CBA-III
1 g/mL

Ag/IH-CBA-III
3 g/mL

Ag/IH-CBA-III
50 g/mL

Ag/Shield-V1
(Mark II)

Ag/Shield-V2
(Mark I)

Assay/Collagen Type

Type 1 VWD Type 2 VWD

Figure 2 von Willebrand factor (vWF) levels (A) and ratio of vWF:antigen (Ag) to vWF:other assay (B) as determined for all test
systems evaluated in the present study and using identical plasma sets derived from type I (asterisks) or type II (open squares
[only type IIA or IIB phenotype evaluated]) von Willebrand disease (vWD). The small horizontal line for each data set represents the
mean value. Ratio value (y-axis in B) set to maximum ratio value of 10. X-axis labels identify each test system. Historic data are the
patients original (initial) data obtained before plasma storage. All other data are test data and ratios determined in the present
study by retesting freshly thawed samples in multiple vWF assays at the same time. The same plasma sample sets (with repeated
testing of some samples) generally were used in each test case. CBA III, collagen-binding (activity) assay using type III collagen;
IH, in house; RCof, ristocetin cofactor. Gradipore, Sydney, Australia; IMMUNO, Heidelberg, Germany; Shield Diagnostics, Dundee,
Scotland.

American Society of Clinical Pathologists Am J Clin Pathol 2000;114:608-618 613

Favaloro / DETECTION OF QUALITATIVE VON WILLEBRAND DISORDER SUBTYPES

Table 2
Summary Correlation Analysis for von Willebrand Factor (vWF):Activity Kit Comparisons*

vWF:Ag vWF:CBA vWF:RCof

r P r P r P

All sample data

vWF:Ag (historic; n = 120) 0.9508 <.0001 0.7177 <.0001 0.8202 <.0001
vWF:CBA (historic; n = 114) 0.6475 <.0001 0.9691 <.0001 0.8651 <.0001
vWF:RCof (n = 113) 0.7591 <.0001 0.8297 <.0001 Perfect line
vWF:Ag (new; n = 120) Perfect line 0.6760 <.0001 0.7624 <.0001
IH vWF:CBA (new; n = 111) 0.6760 <.0001 Perfect line 0.8306 <.0001
IMMUNO vWF:CBA (n = 71) 0.9012 <.0001 0.8441 <.0001 0.8868 <.0001
Gradipore vWF:CBA (n = 40) 0.7537 <.0001 0.9309 <.0001 0.8524 <.0001
Shield Diagnostics ELISA version 1 0.8544 <.0001 0.8698 <.0001 0.8548 <.0001
(Mark II; n = 40)
Shield Diagnostics ELISA version 2 0.9395 <.0001 0.7609 .0003 0.8630 <.0001
(Mark I; n = 40)
vWF:CBA-III (1g/mL; n = 48) 0.6977 <.0001 0.9631 <.0001 0.8485 <.0001
vWF:CBA-III (3 g/mL; n = 73) 0.7154 <.0001 0.9744 <.0001 0.8774 <.0001
vWF:CBA-III (50 g/mL; n = 73) 0.7787 <.0001 0.9769 <.0001 0.9008 <.0001
Type II von Willebrand disease sample data
vWF:Ag (historic; n = 44) 0.8565 <.0001 0.3459 .0215 0.6459 <.0001
vWF:CBA (historic; n = 44) 0.5436 .0001 0.8395 <.0001 0.8590 <.0001
vWF:RCof (n = 41) 0.5380 .0003 0.6643 <.0001 Perfect line
vWF:Ag (new; n = 44) Perfect line 0.4148 .0051 0.5578 .0001
IH vWF:CBA (new; n = 44) 0.4148 .0051 Perfect line 0.6695 <.0001
IMMUNO vWF:CBA (n = 25) 0.7603 <.0001 0.5939 .0017 0.7803 <.0001
Gradipore vWF:CBA (n = 13) 0.7147 .0060 0.9523 <.0001 0.7955 .0002
Shield Diagnostics ELISA version 1 0.8557 .0002 0.8868 <.0001 0.7903 .0003
(Mark II; n = 13)
Shield Diagnostics ELISA version 2 0.9421 <.0001 0.8805 <.0001 0.8363 <.0001
(Mark I; n = 13)
vWF:CBA-III (1 g/mL; n = 18) 0.4913 .0384 0.8643 <.0001 0.6514 .0063
vWF:CBA-III (3 g/mL; n = 30) 0.2076 .2709 0.9857 <.0001 0.6676 .0001
vWF:CBA-III (50 g/mL; n = 30) 0.4714 .0085 0.8017 <.0001 0.7785 <.0001

Ag, antigen; CBA, collagen-binding (activity) assay (type III collagen was evaluated using 3 collagen-coating concentrations: 1, 3, and 50 g/mL; ELISA, enzyme-linked
immunosorbent assay; RCof, ristocetin cofactor.
* vWF:Ag, vWF:CBA, and vWF:RCof headings refer to standard in-house vWF reference assays; n is the number of test sample points available for analysis (mean of triplicate
test readings for each sample tested in each assay); historic refers to previous test data (ie, as obtained for test samples before storage); and new refers to data generated from
the present study. Shield Diagnostics, Dundee, Scotland; IMMUNO, Heidelberg, Germany; Gradipore, Sydney, Australia.

as the alternative functional/activity vWF assay systems. In Discussion

other words, the overlap of data obtained using the
IMMUNO and Shield assays would severely limit their
diagnostic usefulness when attempting to specifically iden-
tify a person with type II vWD for further characterization
of the disorder. The Shield version 1 vWF assay (ie, Mark
II) gave better separation of type I vs type II vWD data than
the original (ie, Mark I) assay.
To more fully evaluate assay usefulness, data from the
present study also were compared with data obtained from
participants of a recent multilaboratory survey11 and by
using 3 patients with well-characterized type II vWD (see
Figure 3 for 2 examples). Again, the greatest discordance
in vWF data between vWF:Ag and functional vWF assays
invariably was observed when using the in-house vWF:CBA
procedures or with the Gradipore vWF:CBA. Relatively
higher vWF values always were obtained with the
IMMUNO vWF:CBA, the Shield ELISAs, and the in-house
type III collagen-based vWF:CBA when collagen was used
at a high (ie, 50 g/mL) concentration.
Type II (ie, IIA, IIB, and IIM) vWD defines qualitative
vWF defects characterized by a discordance in plasma levels
of vWF detected by the vWF:Ag and a functional/activity
vWF assay.3 Accordingly, laboratory testing using combina-
tions of these assays can help identify these forms of vWD
and, thus, guide the need for additional laboratory testing.9
The classic functional vWF test is the vWF:RCof. Unfortu-
nately, as previously reported, use of the vWF:RCof assay,
even in combination with the vWF:Ag, occasionally will
miss type II vWD (especially type IIB vWD), since detected
vWF levels can lie within the normal reference range, either
because of low assay sensitivity to loss of high-molecular-
weight (HMW) vWF or high assay variability.8,11,13
The value of using an alternative functional assay, the
vWF:CBA, has been shown.8,11-13,15,16-21 For example, the
authors laboratory has shown the value of determining the
ratio of vWF:Ag to vWF:CBA to help distinguish type I
from type II vWD, as high ratios (ie, >2.0) generally are

614 Am J Clin Pathol 2000;114:608-618 American Society of Clinical Pathologists

 Coagulation and Transfusion Medicine / ORIGINAL ARTICLE

A B

60 100

80
40

VWF (%)
VWF (%)

60

40
20
20

0 0

S-Ag

S-RCof

S-SE

S-IH-CBA

S-Grad-CBA

IH-CBA

Grad-CBA

IMMUNO-CBA

IH-CBA-III-1

IH-CBA-III-3

IH-CBA-III-50

SE-V1

SE-V2
S-Ag

S-RCof

S-SE

S-IH-CBA

S-Grad-CBA

IH-CBA

Grad-CBA

IMMUNO-CBA

IH-CBA-III-1

IH-CBA-III-3

IH-CBA-III-50

SE-V1

SE-V2

10
Ratio (VWF:Ag/VWF: "Other")

S-Ag/RCof
S-Ag/CBA
8
Ag/IH-CBA
Ag/IMMUNO-CBA
Ag/Grad-CBA
Ag/IH-CBA III-1
6 Ag/IH-CBA III-3
Ag/IH-CBA III-50
Ag/SE-V1
Ag/SE-V2
4

0
S-VWF-4(VWD-2B) S-VWF-10 (VWD-2A)

Assay/Collagen Type

Figure 3 Data from present study compared with those obtained by participants of a multilaboratory evaluation11 and using 2
type II von Willebrand disease plasma samples (1 type IIB [A] and 1 type IIA [B]). The Y-axis shows the level of detected von
Willebrand factor (vWF; percentage of normal) for each sample tested. The X-axis identifies each test group. C, Resultant ratios
for vWF:antigen (Ag) to vWF:other assay for data shown in A and B. The Y-axis shows ratio values set to maximum value of 10.
The X-axis identifies each plasma type (left, type IIB; right, type IIA). Combined method data only shown for survey (S); other
data sets are from the present study. The small horizontal line for each data set represents the mean value. CBA III, collagen-
binding (activity) assay using type III collagen (collagen-coating step used at 3 concentrations: 1, 3, and 50 g/mL); Grad,
Gradipore, Sydney, Australia; IH, in house; IMMUNO, Heidelberg, Germany; RCof, ristocetin cofactor; SE-V1, Shield Diagnostics
ELISA [enzyme-linked immunosorbent assay] kit version 1 (Dundee, Scotland); SE-V2, Shield Diagnostics ELISA kit version 2
(Dundee, Scotland).

consistent with type II vWD. 8,9,11-13,22 ELISA-based vWF defects; the study also compared the assays with alter-
vWF:CBA kits are available commercially from 2 manufac- native methods.
turers. Another potential alternative to vWF:RCof (by All assay methods could detect vWF, as evidenced by
platelet agglutination) also has been described and is based vWF dose-dependent concentration curves (Figure 1). In
on an MAB vWF-capture system. Accordingly, the present general, all methods also could detect type I and type II
study evaluated 4 commercial ELISA-based kit options vWD (Figure 2A), although, in confirmation of previous
representing functional/activity assays for their relative findings,8,11,13 vWF levels detected in type II vWD using the
usefulness for detecting vWD and identifying qualitative vWF:Ag or vWF:RCof assays occasionally lie in the normal

American Society of Clinical Pathologists Am J Clin Pathol 2000;114:608-618 615

Favaloro / DETECTION OF QUALITATIVE VON WILLEBRAND DISORDER SUBTYPES
reference range (Figure 2A), and this could lead to diagnostic
failure to detect these forms of vWD in some test cases
(particularly type IIB vWD). Also in line with previous obser-
vations,8,11,13 use of the vWF:CBA permitted specific identifi-
cation of qualitative vWF defects, evidenced by correspond-
ingly lower vWF:CBA values (compared with vWF:Ag) in
type II vWD (Figure 2A) or higher vWF:Ag to vWF:CBA
ratios (Figure 2B).
Overall best identification of qualitative vWD subtypes
was obtained using the in-house vWF:CBA assays (ie, the
reference method using HORM collagen or using type III
human collagen at a low collagen-coating concentration [ie,
1 or 3 g/mL]). Identification of qualitative vWD subtypes
also was good when using the Gradipore vWF:CBA (also
based on an equine tendon mixed collagen product). The
IMMUNO vWF:CBA (which also uses type III human
collagen), the Shield ELISAs (which use an MAB against
vWF to capture vWF in the assay), and the in-house
vWF:CBA using type III human collagen at a high collagen-
coating concentration (ie, 50 g/mL) were less effective for
identifying qualitative vWD subtypes. Although all could
detect type II vWD (Figure 2A), they each also showed
reduced effectiveness in specific identification of type II
vWD as a qualitative vWF defect (Figure 2B).
This differential finding was despite correlation
analysis, which tended to show good correlation of data
between all assays. Although often used as a means to
compare procedural endpoints for overall similarities, and
although strong correlation often is promoted as good
evidence for equality of measurement, it is obvious from the
present study that good correlation does not necessarily
reflect equality in usefulness. Rather, there is a strong rela-
tionship between the assay readings, as reflected by the
general pattern of data and the fact that all assay methods
measure vWF. Some difference in data points would be
expected in vWF:Ag vs the functional assays for the type II
vWD plasma group, and this was observed (Table 2; thus,
correlation of assay data for type II vWD plasma group is
best for alike assays and, correspondingly, less so for less-
alike assays).
The commercial MAB-based kits seemed to be the least
effective for specifically identifying type II vWD subtypes,
although the recently modified procedure (ie, Mark II)
yielded noticeably better identification compared with the
original assay procedure (ie, Mark I). Limitation in the
ability to specifically identify type II vWD using the
commercial kit method (Mark I assay) has been
reported,11,19,22,26 despite earlier promising studies by the
developing laboratory.23-25 For example, in a multilaboratory
survey,11 the kit method yielded absolute vWF values for
type II vWD tested plasma that tended to be in the high
vWF:RCof range (ie, compared with agglutination method),
616 Am J Clin Pathol 2000;114:608-618
despite also providing substantially lower scatter (ie, reduced
variability than the agglutination method in all test cases). In
addition, Fischer et al19 showed correlation of vWF multimer
size to vWF:CBA and vWF:RCof by agglutination but not to
vWF:Ag or to the commercial Shield vWF-activity assay, and
Preston26 reported a higher median vWF activity level by
users of the Shield ELISA compared with the standard
vWF:RCof agglutination method using a type IIA plasma
sample in a UK NEQAS survey. An explanation for these
apparent contradictions has been proposed.27 Using a panel of
10 locally developed anti-vWF MABs,31,32 the authors labo-
ratory showed that specific identification of type II vWD (as a
qualitative vWF defect) is possible using an MAB-based
ELISA, but identification apparently depends on the overall
characteristics of the assay and the MAB used.27
Because of other independent user feedback, the manu-
facturer of the commercial MAB-based ELISA has modified
the procedure and altered the vWF-detection system from an
HRP-labeled rabbit anti-vWF to an HRP-labeled MAB anti-
vWF.28 The modified assay (Mark II), therefore, uses the
same MAB in the vWF capture and detection steps. The new
method has been reported to correlate better with vWF:RCof
than the original method.28 This revised assay kit was not
available for evaluation in a previous study.27 However, the
present report substantiates that the new method better iden-
tifies type II vWD plasma compared with the original
version. The likely reason for this is the reduced availability
in the revised assay procedure of MAB-binding epitopes for
vWF detection on the smaller vWF multimers (since many
of these epitopes are occupied by the same MAB in the vWF
capture process). Hence, the assay will, to some extent,
detect HMW vWF forms, since these forms have proportion-
ally the greatest number of free/available MAB epitopes for
the vWF-detecting MAB.
In any case, in all studies from the authors laboratory,
no MAB-based vWF-capture system could specifically iden-
tify type II vWD subtypes to the same extent as the
vWF:CBA (neither in-house27 nor either version of the
Shield kit method [present report]). However, although all
vWF:CBA assay systems performed better in this regard
compared with the MAB-based systems, some vWF:CBA
systems were more effective than others.
The overall best identification of type II vWD was
obtained using the in-house reference vWF:CBA (using type
I/III collagen mix product from equine tendon), Gradipore
vWF:CBA (also uses equine tendon-derived collagen), or
using the in-house vWF:CBA methods using type III human
collagen at a relatively low concentration (1 or 3 g/mL,
without covalent linkage). The IMMUNO vWF:CBA
seemed to be the least effective in this regard. The
IMMUNO vWF:CBA uses human type III (placental-
derived) collagen in the ELISA plate coatingvWF capture
American Society of Clinical Pathologists

process. Although the product information sheet does not
describe the collagen-coating conditions, the commercial
ELISA is based on an ELISA process developed from the
work of Fischer et al18,19,33 and Siekmann et al.34,35 These
workers tend to use a type III (placental-derived) human
collagen concentration of approximately 3 g/mL, but they
also use a covalent linkage process to more firmly attach the
collagen to the ELISA plate.
In-house vWF:CBA procedures using a range of type III
coating concentrations also were evaluated in the present
study, as well as previously.22 In the present study, overall
data obtained using the IMMUNO vWF:CBA were similar
to those obtained using a higher (50 g/mL) collagen
concentration without covalent linkage. The authors labora-
tory has previously noted that to be most effective in type II
vWD identification, type III human collagen needs to be
used at a relatively low concentration (1-3 g/mL). 22
Although presumably used at this concentration, the covalent
linkage presumably used for the IMMUNO vWF:CBA
seems to have a somewhat negative effectit makes the
assay system too effective in terms of vWF capture. In other
words, making the system too effective for vWF capture
causes all vWF multimer forms to be captured, and, there-
fore, the assay tends to lose the ability to preferentially detect
or capture only HMW vWF. The findings from the present
study suggest that for the IMMUNO vWF:CBA kit to be
more effective for specifically identifying type II vWD, a
lower collagen concentration may be required or the use of
the covalent linkage step may need revision. This is despite
reports of apparent success in detecting HMW vWF in
plasma and concentrate systems using a covalent linkage
vWF:CBA system.18,19,33-35
Conclusions
All evaluated commercial vWF methods generally were
capable of detecting type I and type II vWD. However, the
commercial MAB-based ELISA systems have only limited
ability to specifically identify type II vWD as qualitative
vWF defects, although the revised method assay performs
noticeably better in this regard compared with the original
assay. vWF:CBA procedures tended to perform better than
commercial MAB-based systems for specific identification
of type II vWD. Overall, the best type II vWD identification
was obtained using the in-house reference vWF:CBA, the
Gradipore vWF:CBA, or the in-house vWF:CBA methods
that use a relatively low concentration of type III human
collagen without covalent linkage. The IMMUNO
vWF:CBA was less successful in this regard, presumably
because the method has overoptimized vWF capture at the
expense of HMW vWF selection. In any case, vWD
American Society of Clinical Pathologists
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
subtypes provisionally detected using any of these assays
should be further characterized as appropriate, using other
methods, as previously suggested.9
From the Department of Haematology, Institute of Clinical
Pathology and Medical Research, Westmead Hospital, Western
Sydney Area Health Service, Westmead, New South Wales,
Australia.
Address reprint requests to Dr Favaloro: Dept of
Haematology, Institute of Clinical Pathology and Medical
Research, Westmead Hospital, WSAHS, Westmead, NSW, 2145,
Australia.
Acknowledgments: I thank all the clinicians who send
plasma samples to our laboratory for analysis, and Mark
Hertzberg, MB BS, PhD(Syd), FRACP, FRCPA, and Jerry Koutts,
MD(Syd), FRACP, FRCPA, for continued support and
encouragement. The technical assistance of Rennie Coombs, who
performed the ristocetin cofactor assays, also is acknowledged.
Finally, the staff of the Parramatta Blood Bank is thanked
profusely for continued assistance.
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