Dr. Donna DiMichele welcomed the audience and outlined the program.

1. Completed/Submitted Reports and Recommendations (2000/01)
Papers submitted to Thrombosis and Haemostasis for peer-review:

The North American Immune Tolerance Registry: Practices, Outcomes, Outcome Predictors. DM DiMichele, B. Kroner

Subcommittee recommendations for ISTH web site publication:

The Design and Analysis of Pharmacokinetics: Studies of Coagulation Factors. M. Lee ,M. Morfini, S. Schulman, J. Ingerslev

Revision of the Protocol Recommended for Studies of Safety from Hepatitis and Other Blood Borne Virus Infections of Clotting Factor Products which are Plasma Derived or Contain Plasma Derived Component. Frank G.H. Hill, Christopher A. Ludlam, Pier M. Mannucci.

2. Notes on the new publications policy
Dr. J. Ingerslev informed on the newly adopted policy for reporting SSC Communications to Thrombosis and Haemostasis. Only original research will be published in full length. All other contributions, including subcommittee reports and recommendations, should be limited to manuscripts of1200 words accompanied by full ISTH web site publication.

3. Registries and Studies in Progress

International F VII deficiency registry

On behalf of Prof G. Mariani and the Registry Steering Committee, Dr. J. Ingerslev presented an update of the International Registry of Congenital FVII Deficiency. This registry now contains 420 patients submitted from 54 centres around the world. Twenty two percent of the subjects have severe deficiency (< 2%) and 52% have FVII levels of 20% or below. The racial background in 85% of pts is Caucasian, with an unexpectedly large distribution of pts from Eastern Europe. A Letter to the Editor was submitted to Blood in an effort to recruit more extensively from the US in an effort to establish more diversity of racial background among registry subjects. Data being collected includes 1) Method of diagnosis including antigen/activity correlation; 2) Characterization of hemorrhagic and thrombotic symptoms; 3) Treatment complications; and 4) Gene mutation analysis. Of note, 23% of patients are a symptomatic. Mucocutaneous bleeding accounted for 59% of the bleeding symptoms. The registry has documented eight thrombotic episodes and no inhibitors. In 210 patients, an assumed causative mutation has been established. Missense mutations represent the majority of these mutations. An analysis of genotype ó phenotype correlation is planned. Enrolment continues with enrolment forms available through Dr Mariani (contact by email).

Registry on FIX Inhibitors and Anaphylaxis in Children

Dr. I. Warrier presented an update of the registry that now contains 77 patients worldwide, 57% of whom have allergic symptoms associated with inhibitor development. In 15/24 patients with a genetic diagnosis, a complete gene deletion was found. The median age at inhibitor development was19.5 months ( 9-156), the median exposure days to F IX was 11 (2-180) and the median peak titer was 30 ( 1-960. ITI had been successful in only 2/19 patients. Nephrotic syndrome was reported in 12 cases, all associated with ITI in patients with allergic manifestations. The renal complications seemed unresponsive to steroids and cyclophosphamide. Enrollment continues with forms available through Dr. Warrier. Prof. Mannucciís protocol for the immunological characterization of these pts is still open, but no new patients have been documented since this study has been established. Family studies were suggested by Dr. Hill. There was a request from Dr. Lee to have this information on the ISTH web site for easy reference. Dr. Warrier agreed to this.

International Registry of Clotting Factor Concentrates

Dr. C. Kasper had written an update of her previous registry. All current manufacturers responded to her request for information on factor VIII and IX concentrates. A paper copy of her registry was distributed to attendees. Dr DiMichele will speak to Dr Kasper about the distribution of this information through the web site in the future.

Gene Therapy Registry.

Dr. K. High presented an overview of ongoing gene therapy trials. Currently, 4000 patients have been treated with gene transfer for genetic diseases with one death reported in a non-hemophilia patient. General information can be obtained from www.wiley.co.uk .

Dr High summarized the results of two clinical safety trials with a B-domain deleted factor VIII gene product and one clinical safety trial with factor IX. In the first FVIII trial sponsored by TKI ( Dr. David Roth, PI), an electroporation cell gene transfer model in skin fibroblasts, resulted in1-2% FVIII expression for < 12 months in 4/6 subjects. The reason for cessation of expression as well as the fate of transfected DNA and cells is not known. The planned enrollment of 6 more patients has already begun.

In the second FVIII trial sponsored by Chiron,13 patients were intravenously infused with a FVIII gene/retroviral vector. The liver was the targeted organ. 6/13 transiently acquired >1% but <2% FVIII activity levels. The trial has been stopped and the data is to be presented at this ISTH meeting.

In a single trial in hemophilia B (Drs High and M.Kay, PIs), AAV/F IX was delivered by multiple intramuscular injections into both quadriceps muscles. FIX levels above baseline with decreased replacement product utilization were observed in 3/8 subjects. In all, 19 hemophilia A pts and 8 hemophilia B pts have been treated with no safety issues identified.

Upcoming trials were discussed. A gutted adenovirus/FVIII trial using a full-length DNA construct (Genstar, Dr. G. White, PI) has just enrolled its first subject. Based on data demonstrating improved expression with liver targeting, an intrahepatic AAV-FIX trial is set to begin later this month. Dr High indicated that the NIH Office of Biotechnology Activities is setting up a registry of all trials. She will explore the possibility of a coordinated gene therapy registry on the ISTH web site subsequent to a meeting with the NIH later this month.

International IT Study.

CRM Hay reported that, despite approved funding by all major manufacturers and ongoing ethical approval of a final protocol in all participating countries, the start of study has been postponed due to a global recombinant and plasma-derived FVIII shortage. A Steering Committee meeting is planned in conjunction with this meeting to further discuss study logistics and a potential start date based on manufacturersí projections.

4.Standardization Issues

Report from the NIBSC/ FDA standardization meeting (June 2001)- T. Barrowcliffe

T. Barrowcliffe reported from the NIBCSC/FDA standardization meeting in London, June 2001, with respect to the following issues:

Concentrate working standards:
A harmonized US/European working standard is still the goal. A report on the new Mega 2 std was presented by Dr Mark Weinstein of the FDA. Full discussion was deferred to Dr Weinsteinís later presentation; however, as discussed at the NIBSC, no decision on the adoption of Mega 2 as a harmonized working standard will be made until after the FDA/CBERís completed report is studied further and any concerns addressed. A decision is expected by next yearís meeting.

Data presented in London continue to demonstrate one stage clotting (OS) and chromogenic (Chrom) assay discrepancies among some concentrates and working standards. When this discrepancy exists, the SSC recommendation to use the chromogenic assay for potency labeling still stands, but is difficult to enforce among manufacturers. Currently, 60% of the manufacturers use the chromogenic assay for labeling.

Concentrate reference standards
The current WHO ref standard 6, the first recombinant, was established in 1999 as the 5^th IS concentrate standard and the 4^th IS plasma standard. Replacement in the near future will be necessary, but the Mega 2 standard is not the likely candidate. At the London meeting, discussion centered on whether the replacement should be recombinant, plasma derived (PD) or both. It is likely that a single standard will continue to be used. A possible candidate could be the FDA ampouled material (N). A decision will be made by the next SSC meeting. A survey of manufacturers indicate that they are using internal stdandards calibrated against either Mega 1 or the WHO reference. Most regulatory agencies are using either Mega 1 or EP 2.

Post Infusion Plasma Measurement
The NIBSC /FDA meeting discussion focused on whether to use a plasma or concentrate standard to assay concentrate in plasma. The merits of the OS vs the Chromogenic assay was also discussed. Dr. Barrowcliffe recommends that further investigation into the reason for discrepancy be done, but that a concentrate standard identical to the infused material be currently used. The practicality of this recommendation was discussed. Dr Mertens noted that a survey of the FVIII concentrate field studies indicates that inter-lab variability is still 10-20% and has not improved since 1995. Lack of compliance with SSC recommendations for the assay is largely responsible. Since concentrate calibration and licensing is the most pressing problem, the proposal from Prof. Mertens was as follows:

1) Use of the infused concentrate as standard should be recommended for manufacturersí PK studies; however, manufacturers must still assay the product according to SSC recommendations which include: a) calibration of their internal standards against a concentrate working or reference standard; b) perform assay according to SSC recommendations; and c) use the chromogenic assay.

2) No recommendation should be made at this time on the standard to be used for the routine assay of concentrate in plasma. Although a suggestion was made to have manufacturers use both OS and chromogenic assays, the committee thought that the data would be too confusing. There was no dissenting opinion on Prof Mertens' proposal. Prof Mertens suggested that the meeting summary be placed on the ISTH web site for easy reference.

The Mega 2 Standard-M. Weinstein

Dr. M. Weinstein spoke to the development of the new Mega 2 concentrate standard. Dr Weinstein reiterated the goals of the new standard which is to develop a uniform international working and reference standard to replace WHO 6 that would demonstrate 1) parallel dose response curves; 2) no OS/ Chromogenic assay discrepancy; and 3) long term stability.

Six candidate plasma and recombinant materials were screened by the FDA and narrowed to two high purity plasma products. They were sent to 18 reference laboratories for assay correlation studies. Candidate A demonstrated a consistent 5-10% increase in OS compared to chromogenic assay results in all labs. Candidate B showed variable OS/chrom assay ratios, but better correlation in aggregate, and was therefore chosen. Vials (100,000) and ampoules(5000) were prepared under different conditions and sent to 38 international labs for assay using local methodology and balanced assay design. The results are as follows: Mega 2: OS= 11.3 u/ml (10.2-12.6) Chrom = 8.6 (8.1-9.4) For Sample N : OS= 10.5u/ml (9.5-11.8) and Chrom = 10.7(10.1-11.3). Questions that remain to be answered include reasons for the Mega 2 assay discrepancy; how the working standard potency will be assigned as well as how the potency assignation will affect vial fill, factor supply and clinical outcomes. Further investigation of these issues will be done by the FDA/CBER and a final report submitted to the NIBSC for further consideration. Dr Weinstein also discussed the pros and cons of US manufacturers reverting to the chromogenic assay for potency labeling

WHO Standard for Factors II, VII, IX and X- E. Gray

E. Gray summarized data from a 19 lab collaborative study to evaluate the 3^rd International Standard candidate (99/826). Inter-lab variability for all factors assayed against both the 2^nd IS standard (CV <4%) and local plasma (CV 6-10%) was excellent. Stability was established by accelerated degradation assay. Potencies/ampoule were assigned as follows: FII 0.91; FVII 1.00, FIX 0.86 and FX 0.93. This proposal was circulated to subcommittee members before the meeting with the majority responding and approving. This standard was approved.

Standardization of inhibitor assays- S. Kitchen

On behalf of Dr. E. Preston, Dr. S. Kitchen reported on the Inhibitor Working Partyís attempt to develop a working inhibitor standard. Six lyophilized candidate materials including 3 patient plasmas, 1 rabbit polyclonal antibody( ab) and 2 monoclonal abs were assayed in 15 labs by OS(8), chromogenic (6) or two stage (1) assays. Wide inter-lab variability was seen for all assays. Chromogenic determinations were 15% lower than OS. Within labs, there was good correlation between patient samples and the rabbit polyclonal (r=0.85) but not between patients and monoclonals (r= 0.2 and 0.02). The working group will pursue further studies to obtain reliable stdandards especially for both very low and high/low responder breakpoint inhibitor assays.

Measurement of FVII following RFVIIA therapy in HA inhibitor patients: impact of the thromboplastins used. E. Preston

Dr. E. Preston reported on a collaborative study performed in 27 European hemophilia centers. Lyophilized plasma samples obtained 15 min and 3 hrs in a single pt following a rFVIIa infusion of 108 micrograms/kg was assayed for FVII activity. The labs employed 23 different instrument/reagent combination methodologies. Inter-lab CVís for both samples were 35.6-40%, although good intra-lab assay correlation was noted (r=0.87). Dr Preston suggested that assay standardization should be pursued if clinicians thought monitoring to be useful. Clinicians suggested some utility in life and limb threatening bleeding if a reliable assay was available. Suggestions on the alternative use of a FVIIA or FVII antigen assay were discussed.

6. FactorVIII Measurement: Is a New Paradigm Possible?

Dr DiMichele opened with an explanation for a timely related discussion of optimal FVIII measurement and dosing. Optimal FVIII measurement was necessary because of 1) non-correlation of current assays; 2) the ultimate need to measure FVIII effect on blood clotting and not "drug levels" in plasma; and 3) emerging new paradigms of coagulation. Optimal FVII dosing was required because clotting factor was proving not to be an inexhaustible resource. Furthermore, an increasing worldwide need for this product necessitated a new cost-effective approach to resource management.

The Role of FVIII in a TF-based model: Relevance of the APTT- K. Mann

Dr Mann clarified the two distinct issues with respect to FVII measurement: 1) assay for potency labeling and 2) assay in the individual that takes into account all aspects of bleeding predictability. Using his in vitro blood coagulation modeling system, Dr Mann illustrated several principles of blood clotting: 1) physiologic reactions are subtle and do not go to completion; 2) initiation phase of clotting is TFóbased and consumes fibrinogen ; 3) the propagation phase generates almost all the thrombin secondary to tenase activation of FXa; 4) 99% of this reaction is invisible to a clot-based assay; and 5) reaction kinetics rather than endpoint is important to the understanding of hemophilic bleeding. His view of the ultimate assay integrates procoagulant and anticoagulant physiology in order to determine the amount of FVIII required for clotting in an individual. Ideally, this model should be adapted to point of care monitoring.

The role of FVIII in FX Activation- P. Fay

Discussing detailed data from his own study of FVIII activation kinetics in a purified system, Dr Fay added that the assay of choice needed to measure the prime role of FVIII in FX activation.

APTT Waveform Analysis: A possible Alternative?- A. Giles

Based on work performed with the waveform APTT on the MDA 180 by Organon Teknika and Drs Shima and Yoshioka, Dr Giles added that there was more to clotting than the endpoint clot. The waveform APTT documents the maximal acceleration of clotting kinetics in a plasma-based optical system. In this system, Drs Shima and Yoshioka noted 5 different patterns among 23 severe HA patients. Re-addition of purified FVIII resulted in a linear correlation between waveform normalization and FVIII activity (r= 0.98). The individual optical responses create the possibility for genotype-phenotype correlation studies.

The Chromogenic Assay- K. Mertens

In a discussion of this assay, Dr Mertens demonstrated that manufacturersí assay recommendations may need to be modified to create optimal activation times necessary for linear kinetics. For this reason, he recommended standardization of the methodology. He reiterated that concentrate PK measurements in plasma serve a different function than the impact of FVIII on clotting. A single assay for both purposes may not be possible.

Discussion- T. Barrowcliffe, P. Lollar, R. Montgomery

A round table discussion emphasized the dual reasons for measuring FVIII; the complexity of that measurement; the need for ongoing study of optimal measurement; and the importance of optimal measurement when using cost effective dosing for bleeding prevention. All participants were encouraged to design and participate in these investigations and to submit data to the next subcommittee meeting.

7. FactorVIII Dosing: Developing Future Strategies

Historical Perspective on Dosing- H. Roberts

H. Roberts reviewed the history of management of bleeding in hemophilia. He suggested that adequate dosing strategies depended on whether treatment or prevention of bleeding was the goal. Historical data suggested that doses of 17 u/kg FVIII could result in breakthrough bleeding and advanced arthropathy. Consequently, minimum dosing strategies have increased over time.

Alternative Dosing Strategies- A. Srivistava

A. Srivastava reported on his extensive experience in India with lower dosing protocols in severe HA patients undergoing primarily orthopedic surgery. His results were as follows: 1) excellent operative hemostasis with target FVIII levels of 60-80%; 2) 14% minor breakthrough bleeding post operatively with target FVIII levels of 17-20%; and 3) total FVIII usage of < 300 u/kg. Based on his experience and a review of the literature, he suggested that there may not necessarily be an inverse correlation between total dose used and bleeding morbidity. A discussion of the generalizability of these strategies ensued.

The Role of TAFI in Hemophilic Bleeding- M. Nesheim

M. Nesheim reviewed the role of TAFI in the attenuation of fibrinolysis through the elimination of the fibin positive feedback loop on conversion of plasminogen to plasmin. He presented data to show that low concentrations of TAFI are sufficient to correct early clot lysis in intrinsic factor- deficient plasma, including FVIII and FIX- deficient plasma. The subcommittee discussed the potential role of fibrinolysis in hemophilic bleeding. The subcommittee debated a potentially greater role for fibrinolytic inhibitors in the optimal treatment of hemorrhage, especially with respect to decreasing the FVIII requirement.

Minimum Effective Dosing: Study Design.- M. van den Berg

On behalf of her collaborator, Dr Gil White, Dr van den Berg presented a rationale and design for a multicenter randomized double-blinded crossover dosing study for hemarthrosis. In this study, 30 patients with severe HA who are >18 years of age and have non-target joint bleeding into knees, would be evaluated for clinical response of acute hemarthrosis to FVIII doses of 30, 20 and 10 u/kg. A salvage dosing schedule is included. Short-term response (24 hours) is to be evaluated by exam and VAS pain score. Long-term response (2 years) is to be measured using clinical and orthopedic joint scores. The subcommittee raised concerns about the delay in outpatient treatment due to double-blinding as well long-term joint morbidity due to potential breakthrough bleeding on low dose regimens. The study was encouraged to proceed upon consideration of the following options: 1) home treatment to prevent delay; 2) comparison of higher dose regimens; 3) potential addition of anti-fibrinolytic therapy to one study arm; 4) a statistical review of cohort size; and 5) a longer follow-up period.

Round Table Discussion: C. Kasper, J. Oldenburg

Dr. Oldenburg (sitting in for Prof Brackmann) discussed the perspective of government-mandated standard of care requirements that would preclude the possibility of a sub-optimal outcome in hemophilia patients. Dr Kasper emphasized the need for reliable cost-effective data to guide the treatment of patients in the developing world. This project will continue in an effort to meet these divergent goals.

Meeting adjourned at 17.30.

Attendance

Most of the time 250-300 people attended the meeting.

Minutes prepared by Drs Ingerslev and DiMichele