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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