International Society Laboratory Hematology Milan, 12-14 May 2016

Current and Emerging Approaches for Assessingvon Willebrand disease (VWD) in 2016

Augusto B. FEDERICI

Hematology and Transfusion Medicine Luigi Sacco University Hospital, University of Milan

augusto.federici@unimi.it

Employment NONE

Research support NONE

Scientific advisory board BAXALTA, CSL-BEHRING, GRIFOLS, KEDRION-LFB, OCTAPHARMA, WERFEN-IL

Consultancy NONE

Speakers bureau BAXALTA, CSL-BEHRING, GRIFOLS, KEDRION-LFB, OCTAPHARMA, WERFEN-IL

Major stockholder NONE

Patents NONE

Honoraria BAXALTA, CSL-BEHRING, GRIFOLS, KEDRION-LFB, OCTAPHARMA, WERFEN-IL

Travel support NONE

Other NONE

DisclosuresA.B. Federici

February 1926E.A. von

WILLEBRAND

1959Nilsson IM et al,

Acta Med Scand; 164:263-

78

1971Zimmerman

TS et al, JCI; 50:244-

54

History of VWD

VWD is the most common inherited bleedingdisorder and is due to quantitative (VWD3 &VWD1) and/or qualitative (VWD2A, VWD2B,VWD2M, VWD2N) defects of VWF: in severe formsof VWD3, VWD1 & VWD2N FVIII is also reduced

Despite the complex and heterogeneous nature ofthe VWF defects, nowadays all VWD types can bemanaged efficiently in most patients.

VWD: Clinical and Lab DiagnosisBackground 2016 (1)

CPlatelet plug formation

BDamaged vessel wall

torque

initialplatelettethering

plateletactivationand adhesion

plateletrolling

activatedaIIbb3

extracellularmatrix

collagenfibrils

A Intact vessel wall

matrixVWF

endothelialcell

platelet

plasmaVWF

GpIba

NonactivatedaIIbb3

How Does VWF Work

• Correct VWD diagnosis and classification cannotbe always available in several Centers to providethe best therapeutic approach.

• Differently from HA easily classified (severe,moderate, mild) by baseline FVIII levels, clinicalseverity of different VWD forms is not welldefined within types so far.

VWD: Clinical and Lab DiagnosisBackground 2016 (2)

List of Clinical and Laboratory ToolsUsed for VWD Diagnosis

Basic Tests Patient & Family HistoryBleeding ScoreBleeding TimePFA 100PTTFVIII:C

Specific tests VWF:AgVWF:RCoVWF:CBVWF:RCo/AgVWF:CB/AgVIII:C/VWF:Ag

Additional tests RIPA testVWF:FVIIIBMultimeric analysisMolecular genetics

More Than One Test Always Needed

Classification of VWD TypesBased on Several Assays

NIH US Guidelines

Heterogeneity of VWD PatientsBased on Cohort Studies

 

VWF:RCo <10 U/dL+ FVIII:C < 20 U/dL

VWF:RCo 10-30 U/dL+ FVIII:C 20-40 U/dL

VWF:RCo 30-50 U/dL+ FVIII:C 40-70 U/dL

VWD Severe Forms:VWD1, VWD2A, VWD3

VWD Moderate FormsVWD1, VWD2B, VWD2M, VWD2N

Bleeders: VWD1 Mild FormsNon bleeders:

Low Levels of VWF

Diagnosed VWD: the tip of the iceberg?

Federici AB et al, Blood 2014; 123: 4037-44.

Heterogeneous VWD Cohort:Italian Registries (RENAWI)

Bleeders versus Non Bleeders

Criteria for Correct Diagnosis(Bleeding History, Low VWF Activity, Inheritance)

Tosetto et al JTH 2006

PlateletGPIba

A1

C C C CA2

SubEndothelium Collagen I and III

Collagen VIHeparinSulphatide

VWF:RCo

VWF:CBA3

ADAMTS 13

W

W

WI:1

III:3 W

I

II

III

Clinical and Lab Diagnosis of VWDOutlines

• Definitions and classification of VWD• Clinical parameters for VWD• First-level laboratory tests• Second-level laboratory tests• Additional and automatic assays • Severe or mild VWD types: outcomes

VWF:RCo(Platelet Aggregometric Method)

Normal Fixed Platelets + Patient Plasma Dilutions + Ristocetin [1 mg/mL]

Multimeric Pattern (2A & 2B)Mutations in the A1 Domain (2M)

Parameters That Influence the VWF:RCo

VWF:Ag

Normal VWD 2A VWD 2B

1 min. 1 min.1 min.

Transmission

Ristocetinmg/ml

VWF:RIPA(Ristocetin Induced Platelets Agglutination)

Platelet Rich Plasma from Patients + RISTOCETIN [0.2-2.0 mg/ml]Ruggeri ZM et al, JCI 1978

VWF Multimeric Analyses(Kindly Provided by U. Budde)

VWF:FVIIIB(Binding Assay - ELISA)

0

750

1500

VWF Immobilized

Bou

ndFV

III:C

Homozygous R854Q

Heterozygous R854Q

Normal

0 750 1500

VWF Pro-PeptideUsefulness of this assay

The assay for VWF pro-peptide measures incirculation the amount of protein cleaved fromPRE-PRO-VWF synthesized in Endothelial Cells

Increased VWF:pp/VWF:Ag ratio identifies thosepatients with shortened VWF survival

Shortened VWF survival can also be observedduring the infusion trial with DDAVP

Ruggeri et al, Blood 1982 Federici AB et al, Blood 2004; 103: 2032-2038

DDAVP Challenge Test: An important assessment at diagnosis

Clinical and Lab Diagnosis of VWDOutlines

• Definitions and classification of VWD• Clinical parameters for VWD• First-level laboratory tests• Second-level laboratory tests• Additional and automatic assays • Severe or mild VWD types: outcomes

VWF:CB(Collagen Binding Activity)

• Evaluates VWF capability to bind to collagen- Mimics VWF interaction with sub-endothelial collagen

matrix at site of vascular injury

• Dependent on VWF multimeric size- Collagen binds more readily with HMWM- Studies show VWF:CB can serve as a surrogate measure

for presence of HMWM

• When tested with VWF:Ag and VWF:RCo can improve differentiation between VWD types 1, 2A, 2B and 2M

VWF:CB(ELISA)

B C D

Add substrateRead OD492 nm

DilutedPlasma

A

Collagen

Anti-VWF-HPR

ConjugatedPolyclonal Antibody

A3VWF

A3VWF

antiVWF

A3VWF

antiVWF

95% type I and 5% type III Collagens (Nycomed-Horm)

Methods not standardized

Type I collagen is more sensitive to discriminate VWD types and is more sensitive to HMWM but it has poor reproducibility

Federici et al, Haematologica 2004

Multimeric PatternMutations in the A1 (2B) A3 (2M)

Parameters That Influence the VWF:CB

Platelet Dependent-VWF Activity(Nomenclature and Methodology)

Bodó et al on behalf of ISTH-SSC-SC on VWF JTH 2014

VWF:RCo(Recent Automated Methods)

Immunoturbidimetric ChemiluminescenceHemosIL VWF:RCo Assay HemosIL AcuStar VWF:RCo Assay

Reaction MechanismLatex particles coated with arecombinant fragment of plateletgp1b, through a monoclonalantibody, which binds VWF in thepresence of ristocetin resulting inagglutination.

Reaction MechanismLatex particles coated with arecombinant fragment of plateletgp1b, through a monoclonalantibody, which binds VWF in thepresence of ristocetin resulting inagglutination.

Gain-of-Function Mutant GPIb-Binding (VWF:GPIbM) Assays

Bodó et al on behalf of ISTH-SSC-SC on VWF JTH 2014

*NotavailableforsaleintheUS.

Flow chart for VWD DiagnosisUsed in Italian Registry

Clinical and Lab Diagnosis of VWDOutlines

• Definitions and classification of VWD• Clinical parameters for VWD• First-level laboratory tests• Second-level laboratory tests• Additional and automatic assays • Severe or mild VWD types: outcomes

Aims of the RENAWI-2

To evaluate the incidence, types and severity of spontaneous bleeding episodes requiring DDAVP and/or VWF concentrates in a large cohort of VWD patients

To characterize bleeding phenotype in different VWD types and to predict clinical outcome in these patients.

Bleeding Phenotype in VWDEvidence-Based Methods

Federici AB et al, Blood 2014; 123: 4037-4044

Restricted Cubic Spline Curve Cox’s Proportional Hazard Model

Bleeding Phenotype in VWD typesType of Bleeding Symptoms

BiologicalResponse in VWD1

No BiologicalResponse in VWD2A

(Ruggeri et al Blood 1982)

DDAVP TreatmentBiological Response to Predict Effective Therapy

Clinical Use of DDAVP According to Clinical Phenotype (BS) of VWD

VWF/FVIII ConcentratesIn VWD3 patients

< 31035

82861

204582

247890

759095

12016585

< 3< 3

7

VWF:RCo =VWF:Ag =

FVIII:C =

Clinical Use of VWF/FVIII Concentrates According to Clinical Phenotype (BS) of VWD

Conclusions of RENAWI-2

The bleeding score (BS) correlates with VWF levels in VWD and helps to predict clinical

outcomes in adult patients with VWD.

Clinical and Lab Diagnosis of VWD Current Perspectives in 2016

• BS and of specific tests for VWF activities should be always used together in Adults to identify VWD patients with bleeding phenotype

• More specific and automatic lab tests should be available in most laboratory world-wide for a rapidVWD diagnosis of bleeding individuals

VWD: Clinical and Lab DiagnosisACKNOWLEDGEMENTS

AB BONOMI Hemophilia Thrombosis Center (Milan):P. BUCCIARELLI, P.M. MANNUCCI, F. PEYVANDI

R. BADER, L. BARONCIANI, M.T. CANCIANI and VWD LAB

Italian Association of Hemophilia Centers (AICE):G. CASTAMAN, M.G. MAZZUCCONI, M. MORFINI,

A. ROCINO, F. RODEGHIERO, M. SCHIAVONI