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Coagulation Disorders
Figure 1: Algorithm for a Laboratory Diagnosis of plasma samples should be thawed to 37ºC before performing the
von Willebrand Disease
diagnostic tests. Special care should be taken to ensure that no
cryoprecipitate is present in the samples; therefore, it must be
Plasma VWF:Ag dissolved before the tests are performed as it will influence the results.
8
Present Absent
Screening Tests
Plasma VWF:RCo versus VWF:Ag
Screening tests for bleeding disorders include a platelet count, a
VWF:CB versus VWF:Ag
Type 3
bleeding time, prothrombin time (PT) and activated partial
Proportionate Discrepant
thromboplastin time (APTT), plasma FVIII levels and the blood group
(0.7–1.2) (<0.7)
of a patient. These tests are usually carried out by a routine
coagulation laboratory.
Type 1
Plasma FVIII versus VWF:Ag
Type 2
VWF:pp versus VWF:Ag
Laboratory Tests
The first line of tests includes the VWF concentration in plasma
Discrepant >2 Proportionate Discrepant
(VWF:Ag), the ristocetin co-factor assay (VWF:RCo) and the collagen
If low levels are detected
FVIII-binding assay
binding assay (VWF:CB). VWF:Ag is measured with an enzyme-linked
immunoabsorbent assay (ELISA). An ELISA plate is coated with a
Type 1
Type 2N
specific rabbit antihuman VWF antibody that captures the VWF to be
With increased
measured. The plasma to be measured is added in 1:50 and 1:100
clearance of VWF
Type 2 dilutions in blocking buffer. Afterwards, a rabbit antihuman VWF
Multimer analysis antibody conjugated to peroxidase is added. This antibody binds to
the remaining free antigenic determinants of VWF, subsequently
HMW multimers
forming a ‘sandwich’. The bound enzyme peroxidase is revealed by
RIPA
Present Absent
its activity in a pre-determined time on the substrate ortho-
phenylenediamine (OPD) in the presence of hydrogen peroxide. After
stopping the reaction with a strong acid, the intensity of the colour
Type 2M Type 2A Type 2B
produced bears a direct relationship to the VWF concentration
Increased Decreased initially present in the plasma sample. A standard curve of calibrated
(0.2–0.8mg/ml) (>1.2mg/ml)
VWF:CB normal
human plasma is used as the standard against which the patient’s
plasma is measured.
RIPA-mixing studies
The RCo assay is performed with formalin-fixed washed platelets in
Plasma defect Platelet defect
an aggregometer with software suited for the test. Formalin-fixed
washed platelets can be obtained commercially or can be self-
Type 2B PT-VWD
prepared from normal platelet-rich plasma (PRP). Washed platelets do
VWF = von Willebrand factor; RCo = ristocetin co-factor; Ag = antigen; CB = collagen-binding;
not agglutinate in the presence of the antibiotic ristocetin unless
pp = propeptide; FVIII = factor VIII; HMW = high-molecular-weight; RIPA = ristocetin-induced normal plasma is added as a source of VWF. The agglutination follows
platelet agglutination; PT = platelet type.
a dose–response curve that is dependent on the amount of plasma
VWF added. Usually, plasma concentrations of 1:2 and 1:4 in Tris-
which may impair its haemostatic function, but also to the influence
exerted by other genes (e.g. those for ABO blood groups).
6
In addition,
many acquired conditions – either physiological (stress, pregnancy) or
pathological (inflammation) – can induce fluctuations in VWF levels).
6 A functional assay that more laboratories
This highly variable clinical picture and the presence of many different
have been starting to use over the last
defects in the VWF molecule complicate the diagnosis of VWD.
6
The
guidelines for diagnosis and treatment of VWD in Italy
7
propose the use
seven years is the collagen-binding assay
of an algorithm (see Figure 1). We adopted and modified these
of von Willebrand factor.
guidelines in our VWD testing facility.
Laboratory Diagnosis of von Willebrand Disease
Sample Collection buffered saline (TBS) are used. A standard curve of calibrated human
Blood samples must be collected into tubes containing 0.105M sodium plasma is used as the standard against which the patient’s plasma
citrate in a ratio of 1:9 with blood. Platelet-poor plasma (PPP) is is measured. Another functional assay that more laboratories have
prepared by centrifugation of whole blood at 2,000g for 20 minutes at been starting to use over the last seven years is the collagen-binding
room temperature. Samples must be stored immediately after assay (CBA) of VWF.
9
The CBA is based on the ability of the HMW
centrifugation in polypropylene tubes at -70ºC until analysed. It is multimers of VWF that preferentially bind to collagen. This is an ELISA-
important to note that a cryoprecipitate may form if plasma samples based assay where dilutions of the patient’s plasma (comparable to
are stored at temperatures over -70ºC. Cryoprecipitate contains large the VWF:Ag 1:50 and 1:100 dilutions) are added to a collagen-coated
quantities of VWF, especially HMW multimers.
8
All tests must be ELISA plate. The type of collagen seems to be important, but
performed on original aliquots that were not previously thawed, and discordance still exists about which type of collagen to use (type 1,
34 EUROPEAN HAEMATOLOGY
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