von Willebrand Factor
DRAFT Subcommittee Minutes
24 July 2011
8:00-12:00
Room A
Chairman: Jeroen Eikenboom (NL)
Co-chairmen: Thomas Abshire (US), Imre Bodo (HU), Jorge DiPaola (US), Emmanuel J. Favaloro (AU), Yoshihiro Fujimura (JP), Paula D. James (CA), Bernhard Lämmle (CH), Reinhard Schneppenheim (DE)
Audience: approximately 250 educational session, 150 business session
Summary of VWF Subcommittee Approvals and Projects
- Project Standardization of VWF propeptide estimation: calibration of reference plasma, assignment of VWFpp value to WHO 6th IS FVIII/VWF Plasma (07/316) and SSC Lot #3. Project will continue to calibrate SSC Lot #4. SCC Official communication will be written.
- ISTH-SSC VWF Online Database (http://www.vwf.group.shef.ac.uk/): a steering committee has been formed this year. A new VWF mutation/polymorphism registry has been created in the Leiden Open Variation Database (LOVD) format. The new registry will go live September 2011. Manuscript in press Goodeve & Hampshire, Semin Thromb Hemost 2011;37:470-9. Ongoing.
- Registry on Acquired Von Willebrand Syndrome (www.intreavws.com): during 2011 the website is being re-organized and updated. Ongoing.
- Registry on platelet-type VWD (www.pt-vwd.org). Ongoing.
- Project “Von Willebrand Factor assays in Von Willebrand disease diagnosis” has ended. An Official Communication of the SSC was published this year: Lee CA et al., Laboratory diagnosis of von Willebrand disease: results from a prospective and blind study in 32 laboratories worldwide using lyophilized plasmas. J Thromb Haemost, 2011;9: 220–222.
- Project “Desmopressin in the management of Von Willebrand disease: biological response versus clinical efficacy”, enrollment closed, data analyzed, manuscript in preparation.
- Project “Standardization of quantitative bleeding scores”. This ISTH-endorsed BAT was published as an Official Communication of the SSC in 2010: Rodeghiero et al., ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. J Thromb Haemost, 2010;8:2063-2065. Work will be continued in the standing committee “Bleeding Assessment Tools”. A proposal was presented for an initial validation study. It has to be decided whether this validation study will be a project within the VWF Subcommittee or will be an activity of the new standing committee “Bleeding Assessment Tool”.
- New ideas:
- development of an international reference preparation for ADAMTS13 activity
- comparative study between different VWF ‘activity assays’
Educational program
The educational program was well attended and got a lot of positive feedback.
Bleeding scores and Bleeding assessment tool
Session Chair: Paula James (CA)
- Update on activities of beeding score working group
Paula James (CA) (on behalf of Alberto Tosetto) provided an update about the activities of the bleeding score working group. This group was established as a collaborative effort between the VWD, Pediatric and Women's Health Issues SSCs and has met regularly since 2008. The initial mandate of the group was to draw on the experience of its members to develop a single bleeding assessment tool (BAT) that could be used in a variety of settings. This ISTH-endorsed BAT was published in the J Thromb Haemost in 2010 and Dr. Barry Coller from Rockefeller University is working with his team to make it web-accessible for research studies. A proposal was presented for an initial validation study. It has to be decided whether this validation study will be a project within the VWF Subcommittee or will be an activity of the new standing committee “Bleeding Assessment Tool”.
- Online bleeding assessment tool
Barry Coller (USA) reported on the Bleeding History Phenotyping Initiative at Rockefeller University and the system they have developed to support the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT). He described: 1) the rationale for an ontology structure, 2) the study of 500 healthy individuals conducted with the Rockefeller questionnaire, 3) the ongoing study of 100 individuals with mild bleeding disorders, 4) the elements of the agreement between Rockefeller and ISTH, 5) how ISTH members can access the ISTH-BAT, 6) the results of the first 50 healthy individual using the ISTH-BAT at Rockefeller, and 7) potential cooperative studies that could be conducted with the ISTH-BAT at multiple sites.
Standardization of assays
Session Chair: Bernhard Lämmle (CH)
- Working party on standardization of VWFpp assays
Tony Hubbard (UK) reported on behalf of the working party on standardization of VWF propeptide assays. Determination of the ratio for VWF propeptide/VWF:antigen (VWFpp/VWF:Ag) is undertaken to identify conditions associated with decreased half-life of VWF in the circulation (eg. type 1 von Willebrand disease with increased clearance, acquired von Willebrand syndrome). Although there is an agreed international unitage (IU) for VWF:Ag, there is no agreed IU for VWFpp. Laboratories therefore rely on local or “unofficial” reference preparations for VWFpp estimation with potential for considerable inter-laboratory variability as well as problems of long-term continuity. The SSC/ISTH Sub-committee on VWF has established a Working Party on Standardization of VWFpp Assays with the following objectives:
- to assess the inter-laboratory variability of VWFpp (and VWF:Ag) estimates
- to calibrate a reference plasma with an agreed unitage for VWFpp
These objectives were addressed through a multi-centre study where 13 laboratories tested two common freeze-dried plasma samples (WHO 6th IS FVIII/VWF Plasma and SSC/ISTH Secondary Coagulation Standard Lot #3) for VWFpp and VWF:Ag relative to their local reference materials.
The inter-laboratory variability for VWFpp estimates was surprisingly low for both common samples (GCVs 8.1%, 8.8%) considering that different local reference materials were used in each laboratory. This variability was reduced even further (GCV 3.0%) when estimates were calculated relative to the same common sample (SSC Lot #3 vs WHO IS) indicating that the different methodologies for VWFpp estimation are in generally good agreement and that a common reference material/unitage could further improve agreement between laboratories. The low inter-laboratory variability for VWFpp estimates also provides the opportunity to assign consensus mean values to both the WHO 6th IS and the SSC Lot #3 of 1.03 IU/ampoule and 1.01 IU/ml respectively. The inter-laboratory variability for VWF:Ag estimates, relative to local reference materials (GCVs 12.2%, 12.0%), was larger than that found for VWFpp estimates despite the availability of the WHO IS for VWF:Ag. This large variability is probably caused by inaccurate calibration of the local reference materials rather than different methodologies since the variability was greatly reduced (GCV 3.5%) when all laboratories compared the same samples (SSC Lot #3 vs WHO IS). This study indicates that increased agreement between laboratories in the estimation of the VWFpp/VWF:Ag ratio may be partially achieved through the establishment of a WHO IS for VWFpp but will also need better harmonisation in the calibration of local references for VWF:Ag.
Project will continue for another year to calibrate SSC Lot #4. SCC Official communication will be written.
- Variation in ADAMTS13 activity assays and the need for standardization
Ian Mackie (UK) reported on the comparison of the commonly used clinical ADAMTS13 activity assays: collagen binding assay (CBA) using full length VWF substrate, in-house Fret-VWF73 assay, commercial Fret-VWF86 and chromogenic VWF73 assays in 159 citrated plasmas from healthy normal subjects and patients being investigated for thrombotic microangiopathies (TMA). They found that:
- Frozen aliquots of pooled normal plasma, QC and normal subject plasmas gave better agreement between assays than TMA samples.
- Lyophilised commercial calibrants gave CBA values (probably due to stabilisers such as HEPES).
- The CBA assay had lower mean values than the peptide substrate assays. Agreement between assays was best in samples with <11% or >55% activity. Only 46% of samples with moderate ADAMTS13 deficiency (11-55%) showed agreement between assays.
- There were some differences between in-house and commercial assays that could have been due to the accuracy of calibration.
Substrate type, denaturing reagents, protease inhibitors, incubation times, dilution factor, diluent, calibrant, gene mutations, antibody subsets, icterus, haemolysis, lipaemia, abnormal amounts of VWF, factor H, and FVIII could impact differently on each assay. It is unclear which assay has the best clinical utility, but it is important that the same assay is used throughout the clinical management of a patient. The development of an International reference preparation and formal EQA schemes are needed.
- A 10 min-assay for ADAMTS13 activity with an automated biochemistry analyzer
Dr S. Kato (JP) presented the results of a newly developed automated ADAMTS13 activity assay that makes use of colloidal gold particles coated with antibodies (either N10 antibody - monoclonal antibody that specifically recognizes Y1605, which is the C-terminal edge residue of the enzymatically cleaved VWF-A2 domain – or anti-GST antibody) and the substrate GST-VWF73-His. The aggregation of the colloidal gold particles is followed as a measure of the level of cleavage of the substrate by ADAMTS13. Assay has very good inter- and intra-assay variability.
- ADAMTS13 activity assays: Comparison of different methods
Johanna Kremer Hovinga (CH) showed that despite improved ADAMTS13 activity assays we still have variation between different ADAMTS13 activity assay results. Discussed assays are: an improved FRETS-VWF73 assay and a multimer degradation assay. A few examples were shown to illustrate what we can learn from discrepant results. She concluded that we don’t need better assays, but that we have to learn what we can learn from discordantADAMTS13 activities.
Based on the results of these presentations on ADAMTS13 activity it was suggested to explore the possibilities for the development of an international reference preparation for ADAMTS13 activity. Johanna Kremer Hovinga (CH) will make a proposal for a study and submit that to the subcommittee.
- Collagen binding assay, current status
Jerry Koutts (AU) gave a short update on the Collagen Binding assay and its use as a supplementary assay for VWF activity. The ristocetin cofactor assay represents the main functional assay in general laboratory use, but suffers from relatively high performance time and complexity, poor reproducibility and poor sensitivity for low levels of VWF. The collagen binding assay substantially reduces the diagnostic error rate in VWD when the usual test panel of VWF:Ag, VWF:RCo and FVIII:C is used. This benefit was originally highlighted by data from the RCPA Haematology QAP and recently confirmed by a NASCOLA study. Standardisation issues are currently noted to hamper the broader utility of the assay and commercial kits differ in efficacy.
- Performance data of a particle enhanced VWF activity assay with no need of ristocetin
Juergen Patzke (DE) presented data on the performance of a new ristocetin-independent VWF activity assay (Not available for sale in the US). Fully automated assay applications on several hemostasis instruments were developed. Reagents are liquid and ready-to-use, the measuring range covers 4-600% for most instruments, the limit of detection is below 2.2 % VWF and Within Device CVs are in general between 2 and 7%. The correlation to the VWF:RCo assay is excellent. All types of von Willebrand disease show a very good comparability of the activity/antigen ratios of the new assay and the VWR:RCo assay.
Based on this presentation it was suggested to make an inventory of all available VWF ‘activity assays’ and to have a comparative study between the different methods. Also nomenclature for the new assays should be decided upon. A potential protocol for such a study will be designed by Juergen Patzke (DE), Reinhard Schneppenheim (DE), Imre Bodo (HU) and Flora Peyvandi (IT) and then submitted to the subcommittee for approval.
VWF and VWD registries
Session Chair: Imre Bodo (HU)
- Platelet Type–VWD registry/database (www.pt-vwd.org/)
Unfortunately, Dr Maha Othma (CA) was unable to attend, but she provided us with the following written update on the Platelet Type–VWD registry/database. An international prospective/retrospective PT-VWD study (Hamilton etal, Thromb haemost 2011) reported that PT-VWD constitutes 15% of diagnosed type 2B VWD worldwide. 24% of cases thought to have type 2B VWD don’t show mutations in either genes (VWF or GP1BA). Clinical and laboratory phenotype assignment remain to be refined worldwide. The PT-VWD registry is available and maintained at www.pt-vwd.org: The registry shows 55 reported cases worldwide, 36 of which are females. To date, 4 mutations reported: three within the VWF binding domain of the GP1BA gene: Gly 233 Val, Met 239 Val, Gly 233 Ser and one ( 27bp deletion) outside this domain. A fifth mutation; Asp 235 Tyr was recently identified by the Iranian Comprehensive Haemophilia Care Centre (personal communications, added to the registry). Flow cytometry test for diagnosis is published. PT-VWD plasma samples are needed for standardization exercises. PT-VWD animal model is available and is helpful to understand the disease pathology as well as phenotype.
- VWF database (www.vwf.group.shef.ac.uk/)
Dan Hampshire (UK) gave an update on the activities regarding the VWF database. Two major alterations have been made to the online locus-specific database for von Willebrand factor (VWFdb) over the last year: 1) A VWFdb steering committee, consisting of 8 members from 6 different countries, has been established (Dan Hampshire (UK), Anne Goodeve (UK), Reinhard Schneppenheim (DE), Paula James (CA), Dan Bellissimo (US), Luciano Baronciani (IT), Pierre Boisseau (FR) and Steve Keeney (UK)). This committee will oversee classification of VWF variants submitted to VWFdb and will recommend enhancements to VWFdb. 2) A new VWF mutation/polymorphism registry has been created in the Leiden Open Variation Database (LOVD) format following steering committee consultation. The new registry will go live in September 2011 and will contain previously unavailable fields for phenotype documentation, along with a “live summary” of sequence variants on the database and will simplify submission of variants to VWFdb. In addition to these two major alterations, several other modifications have been proposed and were discussed.
Manuscript in press Goodeve & Hampshire, Semin Thromb Hemost 2011;37:470-9.
- International registry on acquired von Willebrand syndrome (www.intreavws.com)
Augusto Federici (IT) gave an update on diagnosis and treatment of AVWS. He discussed a recent paper on this (Blood 2011;117:677-6785). During 2011 the website is being re-organized and updated. Ongoing.
Multicenter studies on VWD
Session Chair: Reinhard Schneppenheim (DE)/Jorge DiPaola (USA)
- European Project on type 3 VWD
Augusto Federici (IT) presented the aims of the project on type 3 VWD (acronym of the study 3WINTERS-IPS). To enroll 300 VWD type 3 patients in Europe and Iran. The study will run for 5 year and will include a 2 year follow-up of each patient to evaluate the frequency and risk of bleeding. From all patients plasma and DNA will collected.
- EUVWD Cooperative Group
Ian Peake (UK) summarized the goals and activities of the EUVWD Cooperative group. The group was formed by the MCMDM-1VWD Partners following the formal end of the project in 2006 and is responsible for the data and samples resulting from the MCMDM-1VWD project. To date the group has published 16 papers based on the MCMDM-1VWD data and several papers are in preparation.
A project on a novel GPIb binding assay is underway. Working with Augusto Federici and collaborators in establishing the EU/Iran type 3 VWD study (VWD-3WINTERS-IPS).Collaborating with the NIH funded Zimmerman VWD PPG. Considering producing a Principles of Care of VWD document in collaboration with EAHAD. Current membership: Ian Peake (Co-chair), Giancarlo Casterman (Co-chair), Anne Goodeve (Secretary), Francesco Rodeghiero (past chair), Agnes Veyradier, Alain Gladisseur, Imre Bodo, Jeroen Eikenboom, Erik Berntorp, Augsuto Federici, Flora Peyvandi, Jorgen Ingerslev, Jenny Goudemand, Javier Batlle, Frank Leebeck, Mike Laffan, Pier Mannicci, Reinhard Schneppenheim, Alberto Tosetto, Ulrich Budde.
- Zimmerman Project (ZPMCB-VWD)
Bob Montgomery (USA) reported on the status of recruitment and frequency of mutations observed in the ZPMCB-VWD project. Until now, 570 index cases have been recruited, including all subtypes. Comparable to previous European and Canadian type 1 VWD studies mutations are very likely to be found when VWF levels are below 20 IU/dL. A striking finding was the very high frequency of a type 2N mutation (H817Q) in African-Americans: 14% heterozygotes. The factor VIII binding defect is mild, even in homozygotes. The frequency or the well-known R854Q mutation in Caucasians was 2.5% heterozygotes, confirming earlier European data.
- Potential importance of the CHARGE loci in VWD patients
David Lillicrap (CA) reported on the results of recent genetic analysis of several large type 1 VWD cohorts which suggest that genetic loci other than VWF likely contribute to low plasma VWF levels. These contributions may be as either the primary pathogenic locus or as genetic modifiers of the type 1 phenotype. The CHARGE (Cohorts for Heart and Aging Research in Genome Epidemiology) consortium has recently published the results of their meta-analysis of 5 large genome-wide association studies evaluating genetic associations with plasma levels of FVII, FVIII and VWF. Results on the discovery population of 23,608 were replicated in a second independent population of 7,604. In addition to confirming the role of ABO and the VWF, 5 novel loci were identified in both the discovery and replication studies: STXBP5, SCARA5, STAB2, TC2N and CLEC4M. These results suggest that additional genes associated with both VWF biosynthesis and clearance influence plasma levels of the protein. Studies to further evaluate these associations are now underway.
- VWD International Prophylaxis (VIP) Study
Tom Abshire (USA) reported that there are currently 54 Centers participating in this study, 30 from Europe, 23 from North America and 1 from Asia with 117 patients enrolled in the three arms of the study (64 retrospective, 42 GI natural history and 11 prospective). Initial data for the retrospective study was presented as an oral presentation at ASH, 2010 and a manuscript is in final preparation. For the ASH presentation, there were 42 patients studied (60% type 3 VWD) with equally distributed prophylaxis indications (epistaxis = 10; joint bleeding = 9; GI bleeding = 8; other = 15). The median number of infusions per week = 2 and the median infusion dose = 43 U VWF:RCo/kg. For all indications, annualized bleeding rates decreased from a median of 12 bleeds (pre-prophylaxis) to 3.8 bleeds (during prophylaxis; p < 0.0001). There were two patients who developed an inhibitor; one before initiating prophylaxis and the other while on prophylaxis. The positive effect of prophylaxis on bleeding was similar for both children and adults. The VIP study continues to enroll patients.
- WIN (Willebrand in Netherlands)
Frank Leebeek (NL) reported on the Willebrand in the Netherlands (WiN) study, a nation-wide study of patients with moderate and severe von Willebrand disease (VWD). A total of 806 individuals, both children and adults, have been included. Of these individuals data on bleeding history was obtained by questionnaire. In addition blood was obtained form 649 VWD patients and DNA is available of 725 patients. VWF parameters have been measured in a central laboratory. We recently studied quality of life in both paediatric patients and adults included in the WiN study and observed a significant decrease of QoL in VWD patient compared to reference populations, especially in type 3 patients (De Wee, EM, et al JTH 2010, JTH 2011). In the next years we will investigate the variability in VWF levels and bleeding phenotype in VWD patients. Our hypothesis is that this may in part be determined by genetic variations that influence VWF:Ag levels in healthy individuals, as has recently been discovered by the CHARGE consortium (Smith N, et al. Circulation 2010). Furthermore studies on specific patients groups within the WiN study population (women, children, elderly) will be performed.
