1Revised: mmvi

Von Willebrand Factor (VWF) TestingVon Von WillebrandWillebrand Factor (VWF) TestingFactor (VWF) Testing

Marlies Ledford-Kraemer, MBA, BS, MT(ASCP)SH

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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

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Introduction to VWFIntroduction to VWF

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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)

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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

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VWF Roles in Hemostasis

Monroe DM, et al. ATVB 2006;26:41-8

Secondary Hemostasis

Primary Hemostasis

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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

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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)

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Screening Tests for VWDScreening Tests for VWD

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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

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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.

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“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)

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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)

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Confirmatory Tests for VWDConfirmatory Tests for VWD

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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)

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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

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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

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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.

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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)

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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

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One-Stage FVIII Assay

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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

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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).

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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

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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

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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

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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

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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.

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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.

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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

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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

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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.

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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

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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

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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

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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.

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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)

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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

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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)

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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

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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

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Clinical ExamplesClinical Examples

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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)

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VWD 2A (IIC Miami)

Luminography – 1.2% LGT Gel

Red = Normal PlasmaBlack = IIC Miami Plasma

Densitometric Scan

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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

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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

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