SCIENTIFIC SUBCOMMITTEE SESSION
6 July 2007 Palexpo, Geneva, Switzerland
Factor VIII and IX
Chair: A. Srivastava (India)
Co-Chairs: C. Hay (UK), C. Lee (UK), K. Mertens (The Netherlands), C. Negrier (France), F. Peyvandi (Italy), J. Saint-Remy (Belgium), E. Tuddenham (UK), H. van Den Berg (The Netherlands)
The Chair opened the meeting at 15:45 and welcomed the audience of about 200 attendants. He confirmed that there were no modifications in the agenda and mentioned that all topics of interest could not be included this year due to lack of time.
Completed/Submitted reports and recommendations
In the last year, SSC activities resulted in the following publication in the Journal of Thrombosis and Haemostasis:
Section 1. SSC Working group on Rare Bleeding Disorders. Chair – F. Peyvandi Co-Chair – C. Negrier.
Since its inception in 2004 within the FVIII/IX subcommittee, this SSC working group on "Rare Bleeding Disorders" (RBDs) has attempted to improve our understanding of prevalence, diagnosis and treatments of these diseases by developing the Rare Bleeding Disorders database (www.rbdd.org). Some international data is also available from an annual survey conducted by the World Federation of Hemophilia (WFH) (www.wfh.org). Various national registries have also begun collecting epidemiological data on these conditions ( Swiss, North American, United Kingdom, French, Egyptian and other EMBRO countries, Iranian and Indian).
RBD in North America – M. Soucie / A. Shapiro
Dr. Soucie provided information about rare bleeding disorders in the U.S. This data is collected using a public health surveillance system called the Universal Data Collection (UDC) system. The UDC has a national, IRB-approved protocol and collects standardized clinical data and a blood specimen that is centrally tested for transfusion transmitted viruses. Patients in any of 135 federally funded comprehensive care centers with a factor deficiency (<50% of normal) or VWD are included, with informed consent. Data collected include demographic, clinical and treatment information, and a self-assessed quality of life tool. Since May, 1998, over 20,000 patients have been enrolled. The distribution of disorders among enrolled patients is 59% hemophilia A, 16% hemophilia B, 22% VWD, and 3% other factor deficiencies. Dr. Shapiro the described the efforts that are also underway to establish a Rare Coagulation Disorders Resource Room, a project of the Medical and Scientific Advisory Council (MASAC) of the National Hemophilia Foundation (NHF). This web-based Resource Room will be devoted to a variety of rare blood disorders, each having a separate manuscript covering clinical, laboratory and genetic aspects authored by an expert.
RBD in South America – E. D’Amico:
Dr. D’Amico presented the South American data. Comprising of 12 countries (total population ~390,000,000) ranging from 458,000 to 191,790,900 inhabitants / country, there is very little published data on RBDs from these countries. Dr. D’Amico had conducted a survey among some of the leading centers in these countries and was able to get data from Brazil, Columbia, Panama, Peru (regional) and Venezuela (national). The reported numbers show wide variability in prevalence of these conditions as documented so far. In all this data, shows the presence of 59 fibrinogen, 62 prothrombin, 41 FV, 132 FVII, 78 FX, 164 FXI, 20 FXIII and 25 combined FV/VIII deficiency patients. The most frequent RBDs are FVII and FXI deficiency. There is limited access to laboratory diagnosis of these conditions. A wide range of products ranging from fresh frozen plasma to factor concentrates are used for treatment, though access is variable depending on the country.
RBD - The WFH survey 2006 – P. Bolton-Maggs
Dr. Bolton-Maggs mentioned that the World Federation of Hemophilia (WFH) had been collecting epidemiological data on hemophilia and other bleeding disorders since 1998 from its national member organizations, which now are over 100 and represent >85% of the world’s population. 56 out of the 101 countries that provide data have some form registry. From 2004, countries have been asked to provide information about the rare bleeding disorders in addition to hemophilia and von Willebrand disease. The number of patients with RBDs reported increases every year and currently runs at more than 17,000. Till 2005, the following numbers of patients with RBD had been reported: fibrinogen-599; prothrombin–167; FV-769; FV/VIII-188; FVII-1689; FX-597; FXI-2446; FXIII-435; platelet disorders-2648. There are many unclassified patients as well. The quality of the data is variable since health care standards vary and the registries may be managed by medical or lay people. This data is accessible on www.wfh.org.
SSC Working Group on inherited RBD- Proposed plan of Action: F. Peyvandi
Dr. Peyvandi described the efforts of above mentioned registries and surveys providing data on RBDs (www.wfh.org/2/7/7_0_Link7_GlobalSurvey2005.htm and
www.rbdd.org). The former has information on 6,934 RBD patients in 98 participating countries and the latter has data on 3,017 RBD patients in 64 participating countries. T he prevalence of RBDs are similar in the two data sets. However, in both these, only very basic information is available on each patient. About 50% of this data refers to patients in Europe. As a consequence, a network of 10 European treatment centres has been formed to develop a new and homogeneous communication tool for reporting, managing, editing and viewing information on RBD patients (www.rbdd.eu)
To help focus on the development of the science around each RBD, it has been decided to establish small expert groups to prepare a roadmap for the development of the science of that condition.
Section II. F VIII / IX: Clinical issues – I (Assay/EQA/Phenotype) Co-chairs: C.A. Lee and H.M. van den Berg
Optimizing the 1-stage assay – J. Polgar
Dr. Polgar stated that current factor assays do not optimally cover the clinically relevant range particularly at the lowest levels by a reliable single calibration curve. Further improvement of the one-stage assays would be possible through introduction of a ‘zero calibration point’ and the establishment of calibration curves with an optimization approach. In the optimization approach: A) More than ten mathematical transformations can be used both for time/activity. B) Two curves can create the calibration, by selecting two groups of calibration points. C) Curve fittings can be linear, 2nd or 3rd order polynomials. D) A cut-off point defines the end of one, and the beginning of the next curve on the combined calibration curve with a smooth transition. Factor assays on analyzers, which use calibrations established by an optimization approach, have a wide range for accurate assays (0%-150% activity) with excellent linearity in the calibrated region and increased tolerance for reagent variations. Widespread use of ‘zero calibration points’ and establishing calibration curves by the ‘optimization approach’ could lessen variability in factors results among clinical laboratories.
EQA for tests of global haemostasis : ROTEM and TEG – S Kitchen, DP Kitchen, I Walker
Dr. Kitchen presented data from the UK National External Quality Assessment Scheme ( UK NEQAS) for Blood Coagulation that has assessed the use of lyophilised plasma samples for thromboelastography (TEG) and Rotational thromboelastometry (ROTEM). Lyophilised plasmas from as series of normal subjects and patients with deficiency of FVII, IX or XI were analysed in a single centre. These studies confirmed that some parameters were similar to those obtained for whole blood which is the sample material usually analysed for patient study. After this preliminary study, 2 pilot exercises were performed in which 7-10 ROTEM users and 13-14 TEG users received samples from normal subjects and a patient with severe FXI deficiency. The participant group included expert haemostasis centres and anaesthetists in operating theatres. The Coefficient of Variation for clotting times was between 10 and 121% and some clearly outlying results were observed. The clot firmness measurements showed CVs of 8 -33% between centres and were lower for plasma samples with higher fibrinogen concentrations. Overall our data show that lyophilised plasma samples can be used for EQA of TEG and ROTEM, and the degree of variability observed in 2 exercises suggests that such EQA could be of benefit in identifying outlying results. Further exercises are planned.
EQA for genetic testing of haemophilia – D. Perry
Dr. Perry presented the evolution of the EQAS for genetic testing of hemophilia in the UK. A pilot scheme was set up by NEQAS in 1998-2000 using whole blood for the intron 22 inversion. At that time many laboratories were using southern blotting and only a few long range PCR. In 2003, an advisory group was set up to provide a robust scheme for genetics. It was planned to provide samples, set up immortalised cell lines and set up a scoring scheme which would also depend on the family history. There are now two exercises each year which provide a clinical history and whole blood or DNA samples for analysis. A collaboration has been set up with NIBSC to set up immortalised cell lines. The lyophilised DNA has provided conflicting results but the liquid DNA has performed well. There has been encouragement to laboratories to use standard gene monenclature – the CMGS (Clinical Molecular Genetics Society) system. Participants are required to 1) identify the individual 2) identify the mutation 3) answer the question and 4)be precise and this is the basis of the scoring system. At present the exercises have been based on FVIII but there are plans to move onto FIX and VWF genetic analysis.
Phenotypic heterogeneity of severe hemophilia – Newer players. A. Srivastava
Dr. Srivastava mentioned that clinical observation of minimally treated patients with severe hemophilia suggested that the phenotypic heterogeneity among them exists not only in terms of the frequency of bleeding but also at the level of the inflammatory response in the joint. It was therefore hypothesized that polymorphisms in the genes of hemostatic factors and inflammatory cytokines could both affect clinical phenotype. Based on this hypothesis, patients with severe hemophilia were classified as mild (<5 bleeds/year, <10 WFH clinical and <10 Pettersson radiological score) or severe phenotypes (all others). Of the 114 patients evaluated, 14 were classified mild by these criteria. Among these patients, ‘severe’ mutations (inversions / deletions) (RR: 4.8), FVII 353 polymorphisms (arg/gln-gln/gln; lower levels) (RR: 5.9), protein C 1476 AT/TT (higher levels) (RR: 4.0) and TNF a 308 GA/AA (higher levels) (RR: 3.9) were found to be associated with clinically severe disease. With plausible biological basis for these polymorphisms affecting clinical phenotype, these data suggest that the clinical heterogeneity of severe hemophilia is not only determined by a balance of various coagulation proteins but also by polymorphisms in inflammatory cytokines. This work is on-going.
Section III. Factor III / IX. Inhibitors. Co-Chairs: C.R.M Hay / J. M. Saint-Remy.
This section of the SSC included reports of completed, ongoing and planned studies of the incidence and risk factors for factor VIII inhibitors in PUPS, MTPS and PTPs. Past research has primarily focussed on the incidence and genetic and non-genetic risk factors for inhibitor development in PUPs. However, the risk factors and incidence of inhibitors in PTPs are poorly understood and require further study. Since regulators are using PTPs as their model to test the immunogenicity of new products, it is against this yardstick that these products need to be measured.
Global PTP inhibitor surveillance study: follow-up – D. DiMichele / C.R.M. Hay
Dr. DiMichele outlined the development and growth of the US registry. Data on 529 patients in 9 sites are being retrospectively and prospectively collected and the database is growing. Inhibitor and genotypic testing is being done centrally and 240 patients have been genotyped so far. Dr. Hay described the UK registry, the planned European Adverse event network and the planned German registry. The UK registry has networked all 108 UK haemophilia centres, covering 6500 patients with haemophilia A and a total of >23000 patients with bleeding disorders of all types. Adverse events, such as new inhibitors, and new diagnoses are reported electronically in real time. Most patients have already been genotyped. A wider dataset is about to be collected prospectively to explore risk factors for inhibitor development in PTPs. A European adverse event surveillance system (EUHASS) is the subject of an EU grant application and is hoped to be operational by early next year. This will involve 45 haemophilia centres across Europe serving a total of 14,500 patients. The German Registry (national data) has also been set up and is due to open later this year. It is expected that these four registries will collaborate closely. Harmonization of datasets is being negotiated and it is hoped to present a common dataset in 2008.
International ITI study update – C.R.M. Hay.
Dr Hay reviewed the current status of the International Immune tolerance Study. This is an open randomized comparison of low and high-dose ITI in good risk patients. This study investigates factors influencing outcome, morbidity and cost-effectiveness. The study also forms a framework for several other satellite studies. The power calculation indicates that 90 patients are required to demonstrate a 20% difference between treatment arms and 150 patients to demonstrate equivalence. About 80 patients have been recruited and an interim analysis is expected later this year.
Rodin study: Update – H. M. van den Berg.
Dr. van den Berg explained that this study has been developed to determine the risk factors which cause or prevent inhibitor development in PUPs with severe hemophilia and may have important implications for eventual future prevention of inhibitors in these patients. The RODIN study aims to study potentially modifiable treatment related factors that affect the risk for inhibitors in these patients. It will include a cohort of patients with severe haemophilia who are treated in one of 30 participating European centres for the first 75 exposure days to factor VIII. Clinical data is collected from patients with severe (<1%), moderate (1-5%) and mild (5-25%) haemophilia A or B born after January 1 st, 2000. Data will be collected from patients born between 1-1-2000 and 1-1-2008. In total, 400 patients with severe haemophilia will be collected through the PedNet registry.
Section IV. FVIII / IX Standardization issues. Co-chair: K. Mertens
FVIII collaborative studies: Phase II field study. M. Lee
Dr. Lee reported on the statistical analysis of the SSC 9 th field study. This study included both recombinant and plasma-derived FVIII products. All participants had been asked to perform testing using two different operators on two separate occasions. The aim of this was to find an explanation for the high interlaboratory variation in the field studies, including the 9 th. Multivariate analysis revealed that variability was mainly due to the assay as such, and not to other factors such as operators or days. As in the 8 th SSC study, the main factor introducing variability was the prediluent (FVIII-deficient plasma or buffer) used. This study confirms the previous SSC recommendation that predilution in FVIII deficient plasma should always be applied. In the discussion, Dr. Lee mentioned that the current activities are in full support of the previously published SSC recommendations for the assay of FVIII in concentrates. As such, it seems appropriate to work toward completion of these field studies, if appropriate, by publishing the current conclusions.
First International Standard for Factor VIII inhibitor - update. S. Raut
Dr. Raut provided an update on the collaborative study on proposed reference standard for FVIII inhibitor. Preliminary data of this study had been presented in 2006 and a final report was subsequently distributed to participants, requesting feedback from them. The study highlighted that although one candidate preparation Y (pooled inhibitor patient plasmas- 05/206) had the lowest overall CV (17.7%) with a mean Bethesda titre of 8.2 BU/vial, inter-laboratory variability within the study was relatively high (CVs 17-33%). The intra- laboratory variability also gave high CVs (0.5-36%). Furthermore, when the results were recalculated relative to the 5 candidate preparations, only a slight improvement in inter-laboratory CVs was observed for the patient test plasmas and only relative to sample Y, with minimal further improvement for the Nijmegen modification and hybrid inhibitor methods compared to the classical Bethesda assay when assaying patient / plasma inhibitor samples. Major improvement in the inter-laboratory variability was also observed when assaying residual FVIII activity using the chromogenic assay (CVs: 2.5-20.1%) compared to the one-stage assay (CVs: 17.9-32%), although only 3 laboratories used this method. Following further discussions at a FVIII Inhibitor SWP meeting in Amsterdam (May 2007), it was decided to defer finalization of these results till the reasons for such large inter-laboratory variability have been precisely delineated.
Replacement of the 7th International Standard FVIII concentrate – S. Raut
Dr. Raut further announced the forthcoming replacement of 7 th FVIII concentrate standard. Stocks of the current WHO 7 th IS (99/678) are running low and could be exhausted by 2009. This standard is used for the potency measurement/estimation of FVIII in therapeutic concentrate products, both recombinant / plasma derived FVIII by manufacturers and clinical laboratories. Approximately 600 - 800 ampoules are despatched each year from NIBSC. The current 7 th IS is a plasma derived material. Material (both plasma derived FVIII and recombinant FVIII) for its replacement will be sourced from product manufacturers. The materials to be selected as candidates will be discussed and decided after carrying out in-house comparative assessments (trials fills, accelerated degradation studies and potency estimations). Calibration will be performed by clotting assays and chromogenic assays relative to the current WHO 7 th IS in an international multi-centre study involving manufacturers, clinical laboratories and regulatory authorities. Objective will be to submit to ECBS in October, 2009.
Replacement of the International Standard for Factor VIIa – A. Hubbard
Dr. Hubbard reported that the stocks of the current WHO 1 st IS Factor VIIa concentrate (89/688) are low and a replacement preparation is required. Candidate materials are currently being collected and definitive fills should be completed by the end of 2007. The original calibration of the 1 st IS was performed by one-stage clotting assay, relative to the WHO 1 st IS Factors II, VII, IX, X, plasma (84/665), using the same thromboplastin reagent in all participating laboratories. This was necessary since potency estimations of FVIIa concentrate relative to plasma FVII show considerable variation depending on the thromboplastin reagent used. Since the original thromboplastin reagent used to calibrate the WHO 1 st IS FVIIa concentrate is no longer available it is proposed that calibration should rely on a direct comparison of the proposed 2 nd IS relative to the 1 st IS FVIIa concentrate. This approach is supported by accelerated degradation and real-time stability studies which have indicated that the 1 st IS FVIIa concentrate has not degraded since it was calibrated. Depending on the availability of suitable candidate materials it is planned to complete the multi-centre collaborative study by spring of 2008.
Thrombin generation tests- Report of the 2nd collaborative study – E. Gray
On behalf of the SSC Working Party (WP) on Thrombin Generation Tests, Dr.Gray referred to the fact that this WP was set up in 2004 under Plasma Coagulation Inhibitors subcommittee with the remits to investigate, standardize and validate methodologies for the quantitation of results to facilitate good intra and inter laboratory agreements. As the results from the first study in 2006 indicated, the use of a reference-plasma would lower both intra- and inter-laboratory variability, this second study was carried out to investigate the feasibility of establishing a reference plasma for thrombin generation tests. Six freeze-dried samples including three candidate normal pooled plasmas were sent to 110 laboratories and 128 sets of results were returned for analysis. The majority of the labs used commercial kits (CAT, Dade-Behring-ETP, Technothrombin and In-TDT). Four labs used in-house methods. The results confirmed data from the first study and show that calibration against reference plasma improves intra- and inter- laboratory agreement. All 3 candidates reduce variability, but 2 of these materials were better. The WP is now discussing how a reference plasma should be used and also how it can be established as a SSC reference plasma for thrombin generation tests. The next task for the WP is to investigate the application of thrombin generation tests for use in the study of haemophilic plasma. The WP is requesting collaboration with experts from the FVIII/FIX Subcommittee who are interested in the standardization of thrombin generation tests.
In his concluding remarks, the chairman thanked all the co-chairs, speakers and the audience for their participation and closed the meeting at 19:45 hours.