von willebrand factor (vwf) testingvon willebrand factor ... · – exercise – trauma or surgery...
TRANSCRIPT
1Revised: mmvi
Von Willebrand Factor (VWF) TestingVon Von WillebrandWillebrand Factor (VWF) TestingFactor (VWF) Testing
Marlies Ledford-Kraemer, MBA, BS, MT(ASCP)SH
www.CLOT-ED.com 2
Topics for Discussion
Introduction to VWF
Screening Tests for VWD
Confirmatory Tests for VWD
(FVIII Activity, VWF Antigen, VWF
Function Using Ristocetin, Other VWF
Functions, and VWF Structure)
Clinical Examples
www.CLOT-ED.com 3
Introduction to VWFIntroduction to VWF
www.CLOT-ED.com 4
Von Willebrand Factor History
1926 Described by Eric von Willebrand1928-49 Vessel wall & platelet defect 1952-53 Plasma FVIII deficiency1953-70 Search for VWF1970 FVIII/VWF protein (Zimmerman)1980 Platelets in VWD1985 VWF gene cloned (Sadler)1987 First mutation described (Mannucci & Sadler)
www.CLOT-ED.com 5
VWF Gene / Protein Structure
11-17 18-20 20-28 28 28 28-32 33-34 35-39 40-42 -44 45-48 49-52
S D1 D2 D’ D3 A1 A2 A3 D4 B1-B3 C1 C2 CK
2 3-10
Type 2N
Type 2A(IIC)
Types2A, 2B
Type 2A(IIA)
Type 2M
Type 2A (IID)
Loca
lizat
ion
of V
WD
Type
2 m
utat
ions
Func
tiona
l dom
ains
Y1605-M1606
Multimerization
FVIIIHeparin
GPIbCollagen VI
HeparinCollagen
Types I & III αIIbβ3(GPIIb/IIIa)
Dimerization
Propolypepetide (741 aa) Mature VWF Monomer (2050 aa)Signal(22 aa)
VWF gene, located on chromosome 12, is 178 kB in length
Type 2A(IIE)
RGDS
www.CLOT-ED.com 6
VWF Roles in Hemostasis
Monroe DM, et al. ATVB 2006;26:41-8
Secondary Hemostasis
Primary Hemostasis
www.CLOT-ED.com 7
Von Willebrand Disease (VWD)
Most common bleeding disorder in humansAutosomal inheritance~ 0.8 - 1.3% of population has a detectable, inherited defect in Von Willebrand Factor (VWF)– Low VWF levels, bleeding, and family history (the “holy” three)
Types of bleeding– Mucocutaneous bleeding
• Epistaxis, menorrhagia, ecchymoses & hematomas, gingival and gastrointestinal bleeding
• Results from defect in primary hemostasis
– Soft tissue bleeding (after trauma/injury)• Dental extraction, wounds, post-operatively, post-partum• Results from defect in secondary hemostasis
– VWF is carrier (protector) protein for FVIII
www.CLOT-ED.com 8
VWD CategoriesType 1: partial quantitative deficiency of VWF– Of patients with VWD, 73% have Type 1
Type 2: qualitative deficiency of VWF (21% of VWD patients)– Type 2 variants
• VWD Type 2A (synthesis or stability defect)– Decreased platelet dependent function due to loss of large, functional
polymeric forms (high molecular weight multimers [HMWM])
• VWD Type 2B (gain of function defect)– Increased affinity for platelet GPIb
• VWD Type 2M (“multimer”)– Qualitative defects with decreased platelet dependent function not caused by
loss of HMWM
• VWD Type 2N (FVIII binding defect)– Decreased affinity for FVIII
Type 3: total deficiency of VWF (6% of VWD patients)
www.CLOT-ED.com 9
Screening Tests for VWDScreening Tests for VWD
www.CLOT-ED.com 10
Initial Tests
Personal and family history is vital!Personal and family history is vital!Activated partial thromboplastin time (APTT)– Degree of prolongation depends on FVIII levels (~ <40%)
Bleeding Time– in vivo test – performed directly on patient– Fallen into disrepute and replaced by instruments that
perform “in vitro” bleeding timesPFA closure timePlatelet count– Excludes disorders of primary
hemostasis due to thrombocytopenia that is unrelated to VWD
Platelet
Platelet
www.CLOT-ED.com 11
Assessing VWF Assessing VWF Function Using ShearFunction Using Shear
VWF is an adhesive molecule that exerts its function in areas of the vasculature with high shear forces (stress & rate). Test systems (perfusion chambers) that subject denuded endothelium or collagen to high shear forces are only available in research laboratories. Two devices, available to clinical laboratories, are presented in the following slides.
www.CLOT-ED.com 12
“In vitro” Bleeding Time DeviceDade Behring PFA-100® (Platelet Function Analyzer)
High shear (rates = 5,000 - 6,000 s-1) flow system
Assess platelet-collagen and platelet-platelet interactions
Platelets occlude an aperature within membranes coated with:– Collagen/epinephrine (used for primary screening)
– Collagen/ADP (differentiates dysfunction due to aspirin)
www.CLOT-ED.com 13
Cone and Plate DeviceCitrated whole blood applied to polystyrene plates is exposed toarterial shear rates (1800 seconds-1) for 2 minutes using a cone & plate device– Induces a laminar flow with uniform shear stress over the entire plate
surface that is covered by the rotating cone– System is dependent on plasma VWF with its receptor (GPIb) and
fibrinogen with its receptor (GPIIb/IIIa)• Assess platelet-platelet interactions
– Adherent platelets are stained and percentage of surface covered is noted
Matis Medical Cone and Plate(let) Analyzer (DiaMed Impact)
www.CLOT-ED.com 14
Confirmatory Tests for VWDConfirmatory Tests for VWD
www.CLOT-ED.com 15
Confirmatory ArmamentariumFactor VIII procoagulant activity (FVIII:C)– APTT clot-based or chromogenic methods
Von Willebrand Factor antigen (VWF:Ag)– ELISA or lateximmunoassay (LIA) methods
Von Willebrand Factor (VWF) function– Ristocetin-dependent
• VWF Ristocetin Cofactor activity (VWF:RCo)– Aggregometric, turbidimetric, and ELISA methods
• Ristocetin induced platelet aggregation (RIPA)– Titratable aggregometric method (low shear environment)
– Ristocetin-independent– Collagen binding (VWF:CB)– FVIII binding (VWF:FVIIIB)
VWF structure (VWF multimer analysis)
www.CLOT-ED.com 16
Pre-Analytical ConcernsRefrigerated (2–4o C) transport of citrate-anticoagulatedwhole blood or storage of specimens at this temperature prior to centrifugation & subsequent testing and/or freezing can adversely affect samples to be tested for VWF function (VWF ristocetin cofactor activity and collagen binding) and VWF structure (VWF multimer analysis)– Refrigerated temperatures cause in vitro progressive loss of
high molecular weight multimers (most functional forms) within 3-6 hours
• VWF test results mimic VWD Type 2
– Phenomenon may be caused by cold-induced binding of VWF to platelets or susceptibility of VWF to certain proteases such as leukocyte elastases, plasmin, or calpain
– Reversed by rewarming or retarded if collected in EDTA
www.CLOT-ED.com 17
Reference PlasmaChoice of reference plasma used in preparation of reference curves for FVIII, VWF:Ag, and VWF:RCo affects accuracy– Improper assignment of values for reference plasma can result in
gross over or under estimation of patient FVIII and VWF– Reference material must be traceable to WHO (World Health
Organization) international calibrator plasma
PREPARATION STANDARD MATERIALBlood Coagulation factor VIII and von Willebrand factor, plasma, human. Lyophilized. 0.68 IU / ampoule Factor VIII:C; 0.94 IU/ampoule factor VIII. Antigen; 0.91 IU/ampoule VWF:antigen; 0.78 IU/ampoule VWF:ristocetin cofactor; 0.94 IU/ampoule VWF:collagen binding
5th International Standard, 2003 Human plasma
Blood Coagulation factor VIII concentrate, human. Lyophilized. 11.0 IU / ampoule
7th International Standard, 2003
Recombinant protein
WHO International Biological Reference Preparations
– Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis secondary FVIII plasma standard
• Calibrated by multiple laboratories against WHO standard
www.CLOT-ED.com 18
Assessing FVIII Assessing FVIII ActivityActivity
76.2
71.0
65.7
60.5
48.1
41.4
50.1
53.8
40
45
50
55
60
65
70
75
80
1 10 100Percent Factor Activity
APT
T (s
econ
ds)
Reference curve
38%
Interpolated patient value
50.1
76.2
71.0
65.7
60.5
48.1
41.4
50.1
53.8
40
45
50
55
60
65
70
75
80
1 10 100Percent Factor Activity
APT
T (s
econ
ds)
Reference curve
Reference curve
38%
Interpolated patient value
50.1
38%
Interpolated patient value
50.1
% FVIII SecondsX (log) Y (lin)
1:10 92 41.41:20 46 48.11:40 23 53.81:80 11.5 60.51:160 5.7 65.71:320 2.9 71.01:640 1.4 76.2
Reference Dilution
Example
Patient value is determined by interpolating time, 50.1 seconds using a 1:10 dilution, from the reference curve to yield a FVIII activity of 38%. This indicates that the patient’s FVIII activity level is only 38% that of normal activity.
www.CLOT-ED.com 19
Factor VIII Procoagulant Activity
Measured by clot-based or chromogenic methodsLow plasma levels seen in VWD types 1, 2, and 3– Levels generally mirror those of VWF:Ag except in VWD
Type 2N where FVIII levels are disproportionately lowerGenerally FVIII values are slightly higher than VWF:Agvalues (saturation of VWF sites by normal amount of FVIII)– Patients with VWD Type 3 may show 3-5% FVIII activity
though no VWF:Ag is present (represents free, unbound FVIII)
www.CLOT-ED.com 20
One-Stage Assay for FVIII
Most widely used FVIII assay– First described by Langdell, Wagner, & Brinkhous in 1953
Based on the APTT– Requires both contact activation (APTT reagent activator)
and recalcification (Ca+2 ions)– Design assumes that FVIII supplied by patient plasma is
rate-limiting and that all other components (other clotting factors, phospholipid, and other cofactors) are present at saturating levels
Assay is a “Mixing Study” that uses diluted patient plasma to “correct” the deficiency of FVIII (complete absence) in a FVIII deficient substrate plasma
www.CLOT-ED.com 21
One-Stage FVIII Assay
www.CLOT-ED.com 22
Analytical Variables
Diluent used for making dilutionsComposition of FVIII deficient plasmaInstrument clot detection methodReagent sensitivity to individual factorsNature of reference material used for preparing the reference (calibration) curve (see slide #17)– Impacts FVIII assays irrespective of method (clot-based or
chromogenic)Criteria for acceptability of reference curve
www.CLOT-ED.com 23
Quantitative Quantitative Assessment of Assessment of
VWF AntigenVWF Antigen
Laurell electroimmunoassaywas originally used to quantify VWF antigen. An agarose gel containing anti-VWF antibody is subjected to an electrical current and VWF antigen is precipitated (“rocket”). Since VWF is polymeric, this technique over-estimates protein levels in patients with higher concentrations of smaller multimers (VWD variants). Currently, VWF is quantified by ELISA (enzyme-linked immunosorbent assay) or by LIA (lateximmunoassay).
www.CLOT-ED.com 24
VWF:Ag – ELISA MethodMicrotiter well coated with rabbit anti-human VWFPatient VWF antigen attaches to capture antibodyImmunoconjugated tagged antibody added to form a “sandwich”Bound enzyme is revealed by acting upon a substrate– Color which is released is directly proportional to antigen
concentrationLaboratory sensitivity is to less than 1% VWF:Ag
Y
VWFRabbit anti-human F(ab’)2 VWF immobilized to microtiter plate
Patient plasma containing VWF
Peroxidase conjugated rabbit anti-human VWF detection antibody
AntiAnti--VWFVWF
www.CLOT-ED.com 25
VWF:Ag – LIA MethodParticles coated with polyclonal rabbit anti-human VWF antibodiesVWF antigen is measured by increase in turbidity (increase in absorption or decrease in transmitted light) produced by agglutination of the latex particlesDegree of agglutination is directly proportional to patient VWF antigen (the higher the OD, the greater the amount of antigen)Method limitations– Cloudiness of a plasma
sample leads to under-estimation of VWF level
– Presence of rheumatoid factor leads to over-estimation of VWF level
Linearity– Generally, levels below
10% should be verified by ELISA
www.CLOT-ED.com 26
Regulation of VWF Blood Levels
Levels influenced by– ABO type (affects clearance)– Age– Race
Transient increases noted with beta-adrenergic stimuli– Exercise– Trauma or surgery
Sustained elevation in chronic conditions– Inflammatory states,
malignancy, renal failure, liver disease
123.3
116.9
105.9
74.8
Mean VWF Antigen
63.8
56.8
48.0
35.6
-2SD
A
B
AB
O
ABO Type
www.CLOT-ED.com 27
Assessing VWF Assessing VWF Function Using Function Using
RistocetinRistocetin
Fehlner JR, et al. Proc Natl Acad Sci USA 1972;69:2420-21
Ristocetin (ristomycin) was used as an antibiotic in the late 1950s
Introduced in 1971 as a diagnostic tool for VWD by Firkin & Howard
In 1972 Weiss and colleagues outlined assay for measuring the deficiency of a plasma factor necessary for normal platelet function
Procedure enhanced by Macfarlane in 1975 by stabilizing/fixing the washed platelets with formalin
www.CLOT-ED.com 28
Ristocetin as a Testing AgentRistocetin Induced Platelet
Aggregation (RIPA)Measures ability of patient VWFpatient VWFand patient platelet patient platelet GpIbGpIb to aggregate in the presence of high (1.5 mg/ml) and low concentrations (0.5 mg/ml) of ristocetin
Ristocetin Cofactor (VWF:RCo)
Quantitative assay that determines VWF function by measuring, in patient platelet patient platelet poor plasmapoor plasma, ability of patient patient VWFVWF to agglutinate formalin-fixed platelets in the presence of ristocetin
Assay methodsMacroscopic slide techniqueAggregometryTurbidityELISA
………….Please see lecture entitled Von Willebrand Factor (VWF) Functional Assays for further information
Tracing at right is normal. In VWD Type 2B aggregation would be observed for low dose ristocetin.
www.CLOT-ED.com 29
Assessing VWF Assessing VWF Function Without Function Without
RistocetinRistocetin
The VWF / GPIbinteraction can also be assessed in the absence of ristocetin by either using monoclonal antibodies directed to the glycoprotein Ib (GPIb) binding site on VWF or using other modulators such as botrocetin, a snake venom protein that alters VWF conformation and increases its binding affinity for GPIb.
www.CLOT-ED.com 30
Monoclonal-Based ELISAAssay utilizes monoclonal antibody (MAb RFF-VIII:R/1)
–MAb inhibits ristocetin-induced platelet aggregation and reportedly recognizes a conformationally specific epitope on VWF (molecule is in “active” state) where VWF binds to GPIb
–Subsequent studies have shown that the MAb does not, per se, assess a functional property of VWF (binding to collagen, FVIII, etc) but rather that it is an inhibitor of VWF function
–Appears that MAb, which serves to capture plasma VWF onto microtiterplate, preferentially captures high molecular weight multimers because these forms provide a greater number of antibody binding sites
• Results more closely parallel those of VWF:Ag ELISA than those obtained from aggregometric (functional) method
When initially released in early 1990s, it was thought that the assay could potentially replace aggregometric method
–New version of assay was released in 2000 in which the detectionsystem was modified (HRP-conjugated to monoclonal anti-VWF versus conjugated to polyclonal anti-VWF) to improve sensitivity
www.CLOT-ED.com 31
Monoclonal-Based LIA
Functional immunoassays can be adapted to analyzers by using latex particles in place of microtiter plate wellsParticles are coated with purified anti-VWF mouse monoclonal antibody directed against the GPIb binding site on VWFVWF activity is measured by increase in turbidity (decrease of transmitted light) produced by agglutination of the latex particles– Degree of agglutination is directly
proportional to patient VWF activity (the higher the OD, the greater the activity)
No ristocetin is used in test systemBeckman Coulter
www.CLOT-ED.com 32
Assessing Other Assessing Other Functions of VWFFunctions of VWF
VWF interacts with numerous other proteins. An important localized function, in vivo, is the interaction between VWF released from platelets & endothelial cells and the platelet GPIIb/IIIareceptor, a critical event related to platelet aggregation. Additionally VWF interacts with collagen, FVIII, and heparin.
www.CLOT-ED.com 33
Collagen Binding Assays-1VWF binds via two domains to collagen– Binding by the VWF A1 domain to collagen type VI exposed by
perturbed subendothelium leads to a conformational change in VWF that permits it to bind to GPIb
– Subendothelial collagens type I & III bind to VWF A3 domain
ELISA assays for collagen binding had initially been considered as replacements for the ristocetin cofactor assay (both tests are sensitive to a reduction in high molecular weight multimers)– Can not serve as substitutes since collagen binding assays do not
reflect interaction between VWF and GPIb– Peforming both collagen binding and ristocetin cofactor assays
increases ability to identify VWD Type 2 variants• Aids in the discrimination of VWD types 2A or 2B from Type 2M
www.CLOT-ED.com 34
Collagen Binding Assays-2
Assays have reproducibility issues– Collagens are prone to forming fibrils or aggregates at
neutral pH– Variability in types of collagen and concentrations used for
coating microtiter plates• Equine tendon [type I, type III, or type I/III mixture], bovine
tendon or skin, human placenta [type III, type VI]
Most recent information obtained from the VWF Scientific and Standardization Committee of the ISTH indicates that a collagen mixture of types I/III from equine tendon is the most sensitive to loss of the highest molecular weight multimers
www.CLOT-ED.com 35
FVIII Binding AssayUsed for identifying patients with VWD Type 2NAssay design–Plasma at various dilutions is
added to microtiter wells coated with polyclonal anti-VWF antibodies
–Endogenous FVIII from captured plasma VWF is removed by incubating with CaCl2
–Recombinant FVIII added and amount of bound FVIII quantified by a chromogenic assay for FVIII
–Amounts of immobilized VWF in each well are measured by ELISA
–FVIII binding to VWF is plotted against concentration of VWF bound to microtiter plate well
Gu, J. et al. Blood 1997;89:3263-3269 Normal binding by VWF for FVIII
Patient
www.CLOT-ED.com 36
Assessing VWF Assessing VWF StructureStructure
Qualitative abnormalities of the VWF molecule were initially determined by crossed-immunoelectrophoresis(protein is first separated by molecular charge and then identified immunologically).
In 1980 Ruggeri & Zimmerman and Hoyer & Shainoff published papers describing the use of agarosegels and an ionic detergent to separate the protein according to size and to do so without any purification steps.
www.CLOT-ED.com 37
Study VWF internal structure and distribution of polymeric forms using SDS agarose gel electrophoresis and visualized by autoradiographySeparate protein into high, intermediate, and small molecular weight multimers(MWM)– Loss of high and/or intermediate
molecular weight forms leads to a loss in function (for example, decrease in VWF ristocetincofactor activity)
VWF Multimer Analysis
Gel System: 1.2 % Low Gelling Temperature (LGT) AgaroseNPP = Normal Pooled Plasma; IIC = IIC Miami variant 2A NPP IIC2B 1 IIC
Small MWM (< 5 multimers)
Intermediate MWM (6 - 10 multimers)
High MWM(>10 multimers)
www.CLOT-ED.com 38
Autoradiogram – 1.0% GelLow resolution gel (1.0% LGT agarose)Gel of high porosity which allows all multimers to enter running gel– Internal triplet structure not
discernable
Used to study platelet VWF, post-DDAVP plasma samples or VWF in TTP/HUSAutoradiogram shows a full complement of VWF multimersfrom normal pooled plasma (NPP) and presence of unusually large molecular weight VWF multimers(UL MWH) in plasma from a patient with TTP
NPP NPPNPPTTP
UL MWM
NPP = Normal Pooled Plasma; TTP = Thrombotic Thrombocytopenic Purpura
www.CLOT-ED.com 39
Autoradiogram – 1.2% GelIntermediate resolution gel (1.2% LGT agarose)Gel of intermediate porosity allowing for visualization of internal triplet structure for each multimerUsed for screening since all multimers, except UL MWM, are seen
Discerns normal from pathologic patterns Normal patterns seen in normalcy and VWD types 1, 2M, 2NAbnormal patterns seen in VWD types 2A & 2B, acquired VWD, Pseudo VWD, and improper sample handling
PLT NPP 2A1 IIC 2B 3 NPP = Normal Pooled Plasma; PLT = Platelet (VWF)
www.CLOT-ED.com 40
Autoradiogram – 2.0% GelHigh resolution gel (2.0% LGT agarose)Gel of low porosity– Six to eight of fastest moving,
lowest molecular weight multimers enter running gel
Used to study internal multimer structure– Triplet pattern now seen as a
quintuplet (one central with 2 satellite bands on either side)
Internal multimer patterns differ in various subtypes of VWD such as in Type 2A and Type 2B
IIC NPP NPP 2A
NPP = Normal Pooled Plasma
www.CLOT-ED.com 41
Physiologic Variables Affecting VWF Multimer Analysis
Adapted from Schneppenheim R, Budde U. Semin Hematol 2005;42:15-28.
HypothyroidismDecreased synthesis
Congenital Cardiac Defects, Aortic Stenosis, Endocarditis, Malformation of Vessels, Severe Atherosclerosis, Sickle Cell Disease
Enhanced shear stress
Myeloproliferative Disorders, Enhanced Shear Stress, Uremia, Ciprofloxacin, Primary & Secondary Hyperfibrinolysis, Fibrinolytic Therapy
Increased proteolytic degradation of VWF
Lymphoproliferative Disorders, NeoplasticDiseases, Myeloproliferative Disorders
Adsorption of VWF onto malignant cell clones or other cellular surfaces
Lymphoproliferative Disorders, NeoplasticDiseases, Immunologic Disorders
Specific or non-specific autoantibodiesforming immune complexes that enhance VWF clearance
DisorderPathogenetic Mechanism
www.CLOT-ED.com 42
Clinical ExamplesClinical Examples
www.CLOT-ED.com 43
Diagnostic Application
VWD Type 2A
Ratios of VWF function to antigen– VWF:RCo to VWF:Ag
• Ratio ~1.0 indicates normalcy or VWD Type 1• Ratio < 0.7 indicative of VWD types 2A, 2B, or 2M
– Disparity exists between amount of protein and its functionality– Overestimation of VWF activity will falsely increase ratio
– VWF:CB to VWF:Ag• Ratio < 0.7 in VWD types 2A & 2B• Ratio > 0.7 in VWD Type 2M
Ratios of FVIII activity to VWF antigen– Very low ratios are seen in VWD Type 2N– Ratio ~1.0
• Normalcy• VWD Type 1 due to increased clearance of
FVIII/VWF complex– Ratio >1.0
• Decreased synthesis of VWF (VWD Type 1 due to a VWF null allele)
www.CLOT-ED.com 44
VWD 2A (IIC Miami)
Luminography – 1.2% LGT Gel
Red = Normal PlasmaBlack = IIC Miami Plasma
Densitometric Scan
www.CLOT-ED.com 45
Summary-1Diagnostic testing for VWD should answer the following questions:– How much protein is present?
• Quantify VWF antigen– How well does the protein function?
• Quantify VWF function in order to assess the physiologic interaction between VWF & GPIb or VWF & collagen
– What is the structural appearance of the protein?• Qualitative (can be semi-quantitative) assessment of the protein’s
structure and distribution of its polymeric forms
A composite “picture” that includes answers to these questions is required to make a diagnosis of VWD– Consideration of multiple assays and their relationships to each other
• Ratios comparing VWF antigen to a particular function or variousfunctions to each other
www.CLOT-ED.com 46
Summary-2
VWF test results and diagnosis of VWD are significantly impacted by:– Patient biological variables– Pre-analytical testing issues– Analytical variables
• Calibrators used for establishing reference curves• Laboratory sensitivities of assays (lower limits of detection)
– Post-analytical concerns• Lower limits of reference
intervals used to discriminate normal from abnormal values
• Cut-off limits for various ratios
www.CLOT-ED.com 47
ReferencesBaronciani L, et al. Von Willebrand factor collagen binding assay in von Willebrand disease type 2A, 2B and 2M. J Thromb Haemost 2006, doi: 10.1111/j.1538-7836.2006.02069.xBarrowcliffe TW, et al. Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations. Semin Thromb Hemost2002;28(3):247-56.Budde U, et al. Laboratory diagnosis of congenital von Willebrand disease. Semin Thromb Hemost 2002;28(2):173-90.Cattaneo M, et al. Evaluation of the PFA-100 system in the diagnosis and therapeutic monitoring of patients with von Willebrand disease. Thromb Haemost1999;82(1):35-9.Clinical and Laboratory Standards Institute (CLSI). Assays of Von Willebrand Factor antigen and ristocetin cofactor activity; Approved Guideline H51-A, 2002.Clinical and Laboratory Standards Institute (CLSI). Determination of factor coagulant activities; Approved Guideline H48-A, 1997.Clinical and Laboratory Standards Institute (CLSI). Collection, transport, and processing of blood specimens for testing plasma-based coagulation assays; Approved Guideline H21-A4, 2003.Favaloro EJ, et al. Laboratory testing for von Willebrand's disease: an assessment of current diagnostic practice and efficacy by means of a multi-laboratory survey. RCPA Quality Assurance Program (QAP) in Haematology Haemostasis Scientific Advisory Panel. Thromb Haemost 1999;82(4):1276-82. Favaloro EJ. Collagen binding assay for von Willebrand factor (VWF:CBA): detection of von Willebrands Disease (VWD), and discrimination of VWD subtypes, depends on collagen source. Thromb Haemost 2000;83(1):127-35.Favaloro EJ, et al. Collection and transport of samples for laboratory testing in von Willebrand's disease (VWD): time for a reappraisal? Thromb Haemost2001;86(6):1589-90.Favaloro EJ, et al. Discrimination of von Willebrands disease (VWD) subtypes: direct comparison of von Willebrand factor:collagen binding assay (VWF:CBA) with monoclonal antibody (MAB) based VWF-capture systems. Thromb Haemost 2000;84(4):541-7. Fressinaud E, et al. Screening for von Willebrand disease with a new analyzer using high shear stress: a study of 60 cases. Blood 1998;91(4):1325-31. Gu J, et al. A patient with type 2N von Willebrand disease is heterozygous for a new mutation: Gly22Glu. Demonstration of a defective expression of the second allele by the use of monoclonal antibodies. Blood 1997;89(9):3263-9.ISTH SSC Databse for VWF at http://www.sheffield.ac.uk/vwf/index.htmlLedford MR, et al. New variant of von Willebrand disease type II with markedly increased levels of von Willebrand factor antigen and dominant mode of inheritance: von Willebrand disease type IIC Miami. Blood 1993;82(1):169-75.Ledford-Kraemer MR. The Clotting Times. 2004;4(3):2-9. http://www.clot-ed.com/edit/documents/CTOct2004r.pdfLedford-Kraemer MR. The Clotting Times. 2005;5(2):8-11. http://www.clot-ed.com/edit/documents/CTJul2005.pdfMannucci PM, Tripodi A. Factor VIII clotting activity (Chapter 11). In: Jespersen J, ed. Laboratory Techniques in Thrombosis-A Manual, 2nd ed. Dordrecht: KluwerAcademic Publishers, 2000.Mazurier C. von Willebrand disease masquerading as haemophilia A. Thromb Haemost 1992;67(4):391-6.Montgomery RR. Structure and function of Von Willebrand Factor (Chapter 14). In: Colman RW, ed. Hemostasis and Thrombosis, 4th ed. Philadelphia: LippincottWilliams & Wilkins, 2001.Morales LD, et al. The interaction of von Willebrand factor-A1 domain with collagen: mutation G1324S (type 2M von Willebrand disease) impairs the conformational change in A1 domain induced by collagen. J Thromb Haemost 2006;4(2):417-25.Murdock PJ, et al. von Willebrand factor activity detected in a monoclonal antibody-based ELISA: an alternative to the ristocetin cofactor platelet agglutination assay for diagnostic use. Thromb Haemost 1997;78(4):1272-7.Sadler JE, et al. Impact, diagnosis and treatment of von Willebrand disease. Thromb Haemost 2000;84(2):160-74.Sadler JE. Von Willebrand disease type 1: a diagnosis in search of a disease. Blood 2003;101(6):2089-93. Schneppenheim R, Budde U. Phenotypic and genotypic diagnosis of von Willebrand disease: a 2004 update. Semin Hematol 2005;42(1):15-28.Veyradier A, et al. A new automated method for von Willebrand factor antigen measurement using latex particles. Thromb Haemost 1999;81(2):320-1.Ruggeri ZM. Von Willebrand factor, platelets and endothelial cell interactions. J Thromb Haemost 2003;1(7):1335-42.