Von Willebrand Disease: Challenging Diagnostic Issues

Jorge Di Paola, MD University of Colorado School of Medicine

Children’s Hospital Colorado

FVIII Collagen GP1b α2bβ3

cDNA 0kb 1 2 3 4 5 6 7 8

D1 D2 D’ D3 A1 A2 A3 D4 B C1 C2

Propeptide

Signal peptide Multimerization Dimerization

ADAMTS13

monomer N- -C

dimer

SDS-agarose gel

multimers

>20,000 kDa Courtesy of David Motto

VWF  VWF  Propep*de                  (VWFpp)        

Res*ng  Platelet  

Weibel-­‐Palade  Body   Endothelial  cell  ADAMTS  13  

Clearance  

VWF ADAMTS13 Resting Platelet

Weibel-Palade Body

Endothelial cell Factor VIII Activated Platelet

Platelet Alpha Granule

•  Quantitative Variants

VWF  VWF  Propep*de  (VWFpp)  

Res*ng  Platelet  

Weibel-­‐Palade  Body   Endothelial  cell  

Increased  Clearance  

Decreased  Secre*on  

Platelet  Alpha  Granule  

Type 1 VWD

Type 3 VWD

Type 1C VWD

•  Qualitative variants

Increased susceptibility to ADAMTS13

Increased binding to platelets

Type 2M VWD

Decreased binding to platelets

Decreased binding to FVIII

Type 2B VWD

Type 2A VWD

Type 2N VWD

Decreased Secretion

VWF ADAMTS13 Resting Platelet

Weibel-Palade Body

Endothelial cell Factor VIII Activated Platelet

Platelet Alpha Granule

Case

•  A 13 year old girl is referred to hematology clinic due to excessive menstrual bleeding. She has a history of monthly nose bleeds and easy bruising. Which screening laboratories would you recommend to rule out VWD?

A.  PT, aPTT B.  PT, aPTT, CBC C.  Bleeding time and PFA 100 D.  Available screening tests are not always useful

Screening for VWD (clinical)

•  Importance of personal history of bleeding •  Importance of family history (most cases are

autosomal dominant) •  Use of new clinical tools for screening (such as

questionnaires or bleeding scores) not ready fro prime time yet

Laboratory Screening

•  Complete Blood Count •  PT •  aPTT •  Bleeding Time

•  Platelet Function Analyzer (PFA 100™)

Screening Tests

•  PTT: most times normal unless very low Factor VIII

•  Platelet count: Neither predictive nor diagnostic but may help in determining different subtypes

•  Bleeding time: Low predictive value •  PFA-100: not sensitive for “newcomers”

unless VWF levels < 30 U/dL

Rodgers, R.P. et al. Seminars in Thrombosis and Hemostasis, 16, 1-20 1990 Harrison, P. British Journal of Haematology, 130, 3-10 2005 Quiroga, T. et al. Journal of Thrombosis and Haemostasis, 2: 892-898, 2004

•  The available screening tests are not always useful

•  If personal and family histories of bleeding are strong enough to raise the potential diagnosis of VWD diagnostic labs should be ordered and the patient referred to a hematologist

•  Based on further questioning of the personal and family history you strongly suspect that this 13 year old girl has VWD. Which laboratory tests would you order?

A. Von Willebrand factor antigen (VWF:Ag), Von Willebrand factor ristocetin cofactor activity (VWF: RCo) B. VWF: Ag, VWF:RCo and Factor VIII activity level (FVIII:C) C. VWF:Ag, VWF:RCo, FVIII:C and Multimer analysis D. VWF:Ag, VWF:RCo, FVIII:C, Multimer analysis and gene sequencing of the VWF gene

Case (continued)

Laboratory diagnosis of suspected VWD patients

•  Specific tests   Von Willebrand factor antigen (VWF:Ag)   Von Willebrand factor ristocetin cofactor activity (VWF:

RCo)   Factor VIII activity level (FVIII:C)   Multimer analysis •  Additional tests Low dose ristocetin platelet aggregation (LD RIPA) Collagen binding assay FVIII binding assay Propeptide Assay

Laboratory diagnosis

•  Be aware of preanalytical variables (platelet contamination, refrigeration and heat can affect test results)

•  VWF is an acute phase reactant (therefore it may take more than one measure)

•  Common genetic variants may affect test results but are not clinically significant

Common genetic variants in VWF

Flood et al. Blood 116, 2, 280-6 2010 Flood et al. Blood 117, 6, 67-74 2011 Bellisimo et al. Blood 119, 9, 2135-40 2012

•  3 exon 28 single nucleotide polymorphisms (SNPs), I1380V, N1435S, and D1472H, are associated with a significantly lower VWF:RCo/VWF:Ag ratio

•  These variants do not appear to have any clinical significance

•  New gain-of-function GP1b ELISA may solve this issue

Collagen-Binding Assay

Collagen

VWF

Anti-VWF Ab ELISA Reader

“Not all collagens are created equal” Flood et al. JTH 2012

Laboratory for specific variants of Type 2 VWD

•  Type 2B and Platelet type: Low Dose Ristocetin-induced platelet agglutination assay (RIPA)

•  Platelet type: Ristocetin-induced platelet agglutination assay with normal frozen plasma

•  Von Willebrand factor multimer analysis •  Gene-based diagnosis

Type 2N VWD

•  Suspect in mild hemophilia patients with family history of both male and female affected

•  Symptomatic patients are usually homozygous or compound heterozygous

•  Important to diagnose since patients do not respond to recombinant or monoclonal factor VIII, and should receive concentrates with VWF

Type 2N VWD Assay

Montgomery, Gill and Di Paola Nathan and Oski’s, 2009

 Type  1  VWD      VWF:Ag      VWF:RCo      FVIII:C      Mul*mers                                Normal      

 Type  3  VWD      VWF:Ag        VWF:RCo          FVIII:C    Mul*mers                          Absent  

VWF  VWF  Propep*de  (VWFpp)  

Res*ng  Platelet  

Weibel-­‐Palade  Body  

Increased  Clearance  

Endothelial  cell  

Decreased  Secre*on  

 Type  1C  VWD      VWF:Ag      VWF:RCo    FVIII:C      VWFpp/VWF:Ag  Ra*o        Mul*mers                                          Abnormal                                                                                Higher  MWM        

Platelet  Alpha  Granule  

Increased susceptibility to ADAMTS13

Increased binding to platelets

Type 2M VWD VWF:Ag VWF:RCo FVIII:C Multimers Normal

Decreased binding to platelets

Decreased binding to FVIII

Type 2B VWD VWF:Ag VWF:RCo FVIII:C Multimers Abnormal LD RIPA Enhanced

Type 2A VWD VWF:Ag VWF:RCo FVIII:C Multimers Abnormal

Type 2N VWD VWF:Ag VWF:RCo FVIII:C Multimers Normal

Decreased Secretion

VWF ADAMTS13 Resting Platelet

Weibel-Palade Body

Endothelial cell Factor VIII Activated Platelet

Platelet Alpha Granule

VWD type 1 vs. Low VWF

Basis for the diagnosis of VWD • Mucocutaneous Bleeding

•  Low VWF levels

VWD type 1 vs. Low VWF

•  VWD is characterized for low penetrance and variable expressivity

•  Estimated prevalence in epidemiological studies 1% •  More than 2/3 of the patients are asymptomatic (or

mildly symptomatic) •  Prevalence of symptomatic disease is 1/10.000 •  In large VWD studies less than 60% of families

showed linkage to VWF

Rodeghiero et al. Blood 1987, Werner et al. J Pediatr. 1993

Sadler et al. Thromb Haemost 2000, James et al. Blood 2007

Goodeve et al. 2007

Prevalence of disease

Population Screening Studies

1/100 1/10.000

Patients referred to HTC

Mucocutaneous bleeding

•  Difficult to quantify (subjective most of times) •  Mildly low VWF levels do not predict post-

operative bleeding •  Of the patients diagnosed through population

screening none had significant bleeding a decade later

Biron C, J. Lab. Clin. Med 1999 Castaman G, Thromb. Haemostasis 2000 Miller CH, Blood 1978

VWF levels and VWD

50 <10 40 60

VWF levels

Normal

O

By definition 14% of the Blood Type O individuals have VWF levels <50 U/dL

VWD

Sadler JTH 2005

Diagnostic Levels for VWD:

NHLBI Working Group

Assay “VW Disease” “Possible VWD” or “Low VWF”

VWF:RCo <30 U/dL 30-50 U/dL

VWF:Ag <30 U/dL or normal

30-50 U/dL

Factor VIII â or normal â or normal

NHLBI Von Willebrand’s Disease Working Group Guidelines 2007, Tossetto A, et al J Thromb Haemost 2007

Summary

•  It is critical to make a correct diagnosis of VWD since it has clinical and therapeutic implications

•  New knowledge in genetics and biology of VWF allowed us to improve our understanding of VWD

•  Development of novel assays will improve significantly our ability to diagnose VWD