2011 Factor VIII and IX Minutes

Factor VIII and IX
Subcommittee Minutes

23 July 2011
09:00-13:00
Room A

Chairman: Flora Peyvandi (IT)
Co-Chairmen:  C. Alok Srivastava (IN), Jan Astermark (SE), Kathelijn Fischer (NL), Charles Hay (UK), Claude Negrier (FR), Johannes Oldenburg (DE), Midori Shima (JP), Edward Tuddenham (UK), Leonard Valentino (US)

The meeting started at 9.00 hours on the 23rd of July, 2011 with the Chairman’s welcome speech to over 300 participants. She thanked all Co-chairs and Dr. Alok Srivastava, the previous Chair, for his significant contributions to the activities of the subcommittee.

Welcome and Introduction to the program – F.Peyvandi and A. Srivastava

A summary of the activities of the last 5 years was reported by Dr. Alok Srivastava. Dr. Peyvandi reported the updates of each of the six Projects started in 2009:

• Consensus definitions in Haemophilia: First draft of recommendations is available on ISTH website
• Consensus definitions in Rare Bleeding Disorders: First draft of recommendations is available on ISTH website
• Standardization of methods for performing the Clot Wave Form Analysis
• Standardization of methods for performing the Thrombin Generation Test
• Standardization of methods for performing the Thromboelastogram
• Pharmacokinetics: Manuscript in preparation, will be available on ISTH web-site
• Potency labeling of clotting factor concentrates: Information on the current practice for post-infusion testing is being gathered through a questionnaire circulated to expert clinical laboratories

She mentioned how arrival of different new products (replacement and by-passing therapies) for the treatment of hemophilia A and B to the market necessitates the establishment of a specific program for the future of FVIII and FIX subcommittees. Construction of a well-organized plan in terms of standardization of global assays, assessment of PK, safety and efficacy and development of novel clinical scale (tool) to evaluate therapeutic efficacy of new drugs remains essential. In addition, she highlighted the importance of novel technologies such as large scale genomic sequencing in the field of hemophilia research and care.

The new Project on “Clinical trial design for hemophilia” chaired by Donna DiMichele was also presented. She also described a recent NIH meeting on the role of global assays in hemophilia A. The meeting concluded that collaboration between two scientific committees is recommended (SSC and NIH) in order to avoid duplication of activities and ensure establishment of a single guideline. Both groups have to come up with a specific recommendation on how to use the global assays in hemophilia.

Session 1: 9:00 – 13:00

EDUCATIONAL PROGRAM 

THEME - NEW ERA OF TREAMENT IN HEMOPHILIA
Chairpersons: C. Negrier (France) and K. Fischer (The Netherland)

Jerry Powell - New Products for treatment of haemophilia – changing options

Hemophilia is a rare genetic disease due to deficiency in the function of one of the coagulation factors, resulting in life-threatening spontaneous bleeding when factor activity is less than 1%. Current optimal treatment provides frequent infusions of the missing factor protein to prevent any spontaneous bleeding episodes.  Problems arise because the factor activity still falls to dangerous levels prior to the next infusion, resulting in the risk of spontaneous intracranial hemorrhage and permanent neurological damage, and often intravenous access devices are required to sustain the frequent intravenous infusions, especially in small children, with medical complications of sepsis and thrombosis.  In addition, development of neutralizing inhibitor formation remains a problem.  Several approaches are in development to enhance the duration of efficacy of infused clotting factors.  The first to complete clinical trial was pegylated liposome FVIII. Approaches to increase the half- life of infused clotting factor are currently exploring several innovative changes to the clotting factor, including:  1. fusion of the factor molecule with a portion of the immunoglobulin molecule, 2. fusion of the factor molecule with albumin, 3. addition of site specific polyethylene glycol moieties to the native factor molecule, and 4. modification of sialic acid portions of the factor molecule.  Each of these approaches has promising pre-clinical data for increased half- life and corresponding prevention of bleeding in animal models. A completely different approach to preventing bleeding in hemophilia involves inhibition of the tissue factor pathway inhibitor, potentially allowing the coagulation cascade to bypass the need for clotting FVIII or IX, and activate FX directly. Key questions, for any of these innovative approaches, regarding which is more effective to prevent bleeding in different clinical settings will be addressed in current and planned clinical trials.  Important questions include which of these new factor products will prove most effective for small children, for prevention of spontaneous bleeding in active adults, or in preventing bleeding during surgery; what are potential benefits: avoid post-surgery bleeding or thrombosis episodes. Although no product specific side effects are anticipated, the clinical trials will need to monitor for any unexpected interactions or altered metabolism with long term administration of these modified factor products.  As the clinical trials progress, the other question will be whether any new product can reduce the rate of inhibitor development.  For that answer we can only proceed thoughtfully through clinical trials. Further progress will depend on close interactions between the developers of the new clotting factors and the community of hemophilia patients in order to complete the critical clinical trials to address which way forward is best for hemophilia.

DISCUSSION: Author’s personal review: exposure of more than nine days might be enough for the inhibitor development instead of 50 ED. This was discussed with the audience and the author mentioned that this is his personal experience and not based on the evidence-based data.

Elena Santagostino - Evaluation of safety and efficacy of new products (clinical and laboratory problems)

Recombinant DNA technology and bioengineering strategies have produced novel hemophilia therapeutics with improved functional properties such as increased potency, resistance to inactivation, prolonged plasma half-lives and reduced immunogenicity. In addition to these products developed to facilitate replacement therapy and prophylaxis, other non-replacement, haemostatic agents are also advancing from preclinical to clinical trials.

The clinical investigation plans currently in place to assess the safety and the efficacy of new products for patients with hemophilia have been often discussed because a more uniform standard is still needed between studies conducted in the USA and in Europe, particularly taking into account the body of data required in the context of this rare disease. The application for marketing authorization of new products with modified biological properties poses a number of additional challenges to regulatory agencies, manufacturers, physicians and patients.

Since pharmacokinetic (PK) data are considered the most important surrogate endpoints for the efficacy of a new replacement agent, these should be provided using the same functional assay for analysis of the patient’s plasma and the product. The same assay should also be used for PK studies comparing the “new” with the “old” product.

The clinical investigation plans have been designed according to a stepwise approach in order to involve in specific studies an increasing number of previously treated adults, followed by paediatric patients and, finally, by previously untreated patients (PUPs). PK, safety and efficacy data must be available in a relevant proportion of study subjects in order to start the next trial. This design offers obvious advantages in terms of safety assessment but represents a challenge with respect to the small size of hemophilia population and the demanding schedule of visits and blood drawing. Furthermore, the application of this plan to the investigation of long-acting products raises a number of challenges, i.e., the prolonged PK assessments, the need for a wash-out period for PK studies and inhibitor testing and the time needed to complete the follow-up period (at least 50 exposure days). Even the approved concept of exposure day used to define the schedule of inhibitor testing could become a matter of discussion in the clinical setting of treatment with a long-acting product.

The efficacy evaluation of the product poses also additional challenges: other than PK studies, clinical response should be analyzed, as well as the choice of the functional assays to test the product and the patient’s plasma need particular attention.

In the light of these complex issues, what seems predictable is that the marketing authorization will be delayed for both, adults (waiting for the study completion in all age groups) and PUPs (waiting for the indication after the completion of the specific, post-marketing study). Paediatric patients and PUPs are few and have a limited compliance to demanding study procedures; however they represent the target population for these new products promising easier prophylaxis with less frequent infusions. Post-marketing surveillance through international registries and harmonized data sets could facilitate the collection of high-quality data from a large patient sample as it is needed to exclude altered immunogenicity and to confirm long-term safety of new therapeutics for hemophilia treatment.

In conclusion, harmonization of study design is needed to achieve sufficient data of body clinical data.

DISCUSSION: A prolonged follow-up is mandatory, in particular for new molecules. At the same time FDA and EMEA should share schemes and rules with the help of clinicians providing an assessment scheme for clinical aspects and PK.

FDA AND EMEA REGULATORIES: CLINICAL TRIALS REQUIREMENTS

Nisha Jain - Clinical Trial Designs for Factor VIII and IX Products - FDA

Treatment of Hemophilia A and B has evolved over years: from blood transfusions in 1800s, cryoprecipitate in early 1900s, human plasma derived concentrates in mid 1900s, recombinant factors in late 1900s to modified recombinant factors in 2000s. Over the years safety of the products for treatment of hemophilia A and B has improved. The risk of development of inhibitory antibodies and lack of adherence to prophylaxis regimens because of frequent infusions still remains the main challenge in the treatment of hemophilia patients. The modified FVIII and FIX products are bioengineered to improve the biosynthesis and secretion of the FVIII/FIX molecule, improve the functional activity, extend the half-life and reduce neo-antigenicity. The half-life of currently available FVIII and FIX products is less than 20 hours. Modified FVIII/IX products currently under development exploit several strategies to extend the half-lives such as PEGylation, albumin fusion and Fc fusion.  Licensure of these modified products poses some challenges: what criteria should be used to evaluate efficacy of these products?, should the criteria for safety evaluation for inhibitor development be different than what is currently accepted for the licensed products, can  the currently available standard potency assays  be used to  establish potency of these products. If the dosing frequency changes are substantial for these products an adequate massive education program for both patients and healthcare professionals needs to be launched. These products may provide advantage over the currently licensed products by decreased frequency of dosing because of prolonged half-life. However, safety and efficacy data are needed in order to determine the advantages of these products over currently licensed products.

Anneliese Hilger - Clinical trial requirements for FVIII/FIX – EU-regulatory perspective

The EU-requirements on clinical development for FVIII and FIX products are laid down in guidelines and core Summary of Product Characteristics. The guidelines cover clinical investigations to be conducted pre- and post-marketing authorisation (with differences on patients number requested for each group - >12y, 6-12y, 0-6y - for FVIII and FIX). Guidance is also provided for authorised products where a significant change in the manufacturing process has been made. Clinical trial data, addressing efficacy and safety are required in patients of all age groups for an application for a marketing authorisation. In addition, depending on the type of factor product (e.g. novel protein modifications) studies in previously untreated patients should be performed to investigate efficacy and safety in this specific patient population. In view of the limited availability of patients suffering from haemophilia, data from pre-authorisation studies only are considered insufficient to estimate all aspects of therapy with FVIII/IX products, especially with respect to immunogenicity. Therefore, to collect additional clinical data and to ensure consistency in the long-term between the outcome from pre-authorisation clinical studies and from routine use, a post-marketing investigation should be performed. The clinical development for FVIII/IX products should follow a stepwise approach in order to have some experience in adults and older children before investigating younger children. The clinical investigation in children needs to be supported by an approved paediatric investigation plan. The guidelines exist since more than 10 years and have been recently revised to fulfil progressing legal, scientific and regulatory requirements. Guidelines could be finding soon (www.ema.europa.eu).

DISCUSSION on both regulatory presentations: It was discussed whether there could be some problems with study feasibility, in particular with regard to the required 50 PTP children at time of submission in Europe. A. Hilger stated during the discussion that this is a legal requirement.
Is there any estimation of the number of required patients for all upcoming clinical trials? Do we need to make a priority list? Regulators answered that they were following the current guidelines / requirements but they also acknowledged that while the principles of safety and efficacy requirements cannot change the further development of specific requirements e.g. with regard to numbers of patients and design of clinical trials can be discussed. Some of these issues will be taken up by the Clinical Trail design project group.

BUSINESS PROGRAM

Section 1. Rare Bleeding Disorders (RBDs)
Chairpersons: F. Peyvandi (Italy) and A. Srivastava (India)

Flora Peyvandi - European network on RBDs (EN-RBD): what has been obtained and next steps

Rare Bleeding Disorders (RBDs) include the inherited deficiencies of such coagulation factors as fibrinogen, factor (F)II, FV, FV+FVIII, FVII, FX, FXI and FXIII, and are usually transmitted in the autosomal recessive manner and due to their rarity, sometimes present significant difficulties in diagnosis and treatment. Their frequency in Europe is from 1:500.000 to 1:2 million in the general population but it this increasing due to the high rate of immigration from the Middle East and North Africa where the incidence is significantly higher. The limited available data make it difficult to draw an exact picture of each single RBD. The relationship between laboratory phenotype and bleeding severity in patients with RBDs was explored. Data from 592 patients in the EN-RBD were retrospectively collected over three years. Clinical bleeding episodes were classified into four categories according to severity (ranging from asymptomatic to severe bleedings – grade III). In 513 patients, a linear regression analysis showed that Fibrinogen, FV+III, FX, and FXIII  had the best linear correlation between activity level and clinical severity; FV and FVII deficiencies showed a good correlation whereas FXI deficiency showed no such correlation at all. For FII, number of patients was too small to draw any correlation. Our study remains the first to classify RBDs on the basis of clinical and laboratory parameters, and to highlight significant correlations between the laboratory phenotype and clinical bleeding severity. The main limitation of our study is the retrospective nature of data collection. The observed heterogeneity between different RBDs underlines the need for a prospective data gathering tool that allows for adequate assessment of individual RBDs. Undertaking such a task is one of the future aims of the EN-RBD.

DISCUSSION: Are the categories of bleeding (asymptomatic, grade I, II and III) the criteria recommended for classification of patients? Other than that a bleeding score has been developed, but is currently under validation (the bleeding score developed for VWD1 was not suitable for RBDs).

Mark van Geffen - Nijmegen thrombin and plasmin generation assay in RBDs

Rare bleeding disorders (RBDs) are a heterogeneous group of diseases with varying bleeding tendency, only partially explained by their laboratory phenotype. We have performed simultaneous thrombin and plasmin generation measurements on 41 patients affected with RBDs to investigate whether parameters of the NHA were sensitive to bleeding tendency. In individual groups our analysis showed for patients with prothrombin, FV and FX deficiency and major bleedings an area under the curve (AUC) below 20%. FVII deficient patients had a prolonged thrombin lag-time ratio of 1.6±0.2 (P<0.05) and normal AUC (92 -125%). FXIII deficient plasmas resulted in a reduced thrombin peak height of 59±13% (P<0.05) and normal AUC (90±14%) and afibrinogenemic patients demonstrated plasmin generation of 2-29% of normal. To demonstrate sensitivity of the NHA in this cohort, patients were divided based on their bleeding tendency (major or minor bleeding) and whether their coagulation factor deficiency belonged to the common (FII, FV and FX deficiency) or the non-common (FVII, Fg and FXIII deficiency) pathway. Thrombin lag-time ratio (P<0.001), thrombin peak-time ratio (P<0.01), thrombin peak height (P=0.01), AUC (P<0.0001), plasmin peak height (P<0.05) and plasmin potential (P<0.05) demonstrated significant differences between these groups whereas the FLT ratio did not show any significance. These results provide information over thrombin and plasmin generation phenotype and some NHA parameters show sensitivity to bleeding tendency in the various groups. In conclusion, these results could contribute to more effective clinical management for each individual group.

DISCUSSION: By this new method, only graphs are shown as results and no number or ranges are given. How could this be handled? Enlarging numbers could help in analysis of RBDs by NHA assay.

Clinical trials in RBDs:

Sigurd Knaub - Clinical development plan of a double-virus inactivated fibrinogen concentrate in patients with hereditary fibrinogen deficiency – EU and US requirements

Hereditary fibrinogen deficiencies are rare and present clinically as afibrinogenaemia or hypofibrinogenaemia (type I deficiencies, i.e. quantitative defects), or dysfibrinogenaemia (type II deficiencies, i.e. qualitative defects). Afibrinogenaemia and hypofibrinogenaemia are predominantly associated with bleeding symptoms, while dysfibrinogenaemia may be clinically silent or associated with a mix of bleeding and thrombosis. Plasma-derived fibrinogen concentrate, cryoprecipitate or fresh frozen plasma (FFP) are effective for treating bleeding associated with congenital fibrinogen deficiencies; plasma-derived fibrinogen concentrate is the treatment of choice. Octapharma® is developing Octafibrin – a highly purified, lyophilised, human plasma fibrinogen concentrate for the treatment of congenital fibrinogen deficiencies. Octafibrin if formulated without added albumin and the purification process used in the manufacturing includes two separate virus inactivation/removal steps. A solvent/detergent (S/D) treatment step inactivates transfusion-relevant enveloped viruses and a nanofiltration step, using a PlanovaTM 20 nm nanofilter, is effectively removing enveloped and non-enveloped viruses. None of these steps carry the risk of denaturing plasma proteins. There are no specific guidelines for the clinical development of fibrinogen concentrates in the proposed indication from either the EU or USA. The clinical program for evaluating the efficacy and safety of Octafibrin has been based on the “Guidelines on the Clinical Investigation of Human Plasma-Derived and Recombinant Factor IX products and discussed with an EU Regulatory Agency and FDA. The clinical development program for Octafibrin in patients with hereditary fibrinogen deficiency includes a pharmacokinetic (PK) study (FORMA-01), followed by two efficacy studies (FORMA-02 and FORMA-03). FORMA-01 is a phase II comparative study (with Haemocomplettan® P/RiaSTAPTM as the comparator) investigating PK, efficacy (laboratory endpoints) and safety of Octafibrin in patients with congenital fibrinogen deficiencies and will be performed both in the US and Europe. FORMA-02, an open, uncontrolled phase III efficacy study will document efficacy and safety of Octafibrin in patients with acute or traumatic bleeding. Data from FORMA-01 and interim data from FORMA-02 are required for EU registration. FORMA-03 will compare the efficacy and safety of Octafibrin with Haemocomplettan® P/RiaSTAPTM for on-demand treatment of acute bleeding. FORMA-03 is a phase III study that will be undertaken to fulfil FDA post-licensure requirements. Requirements for the clinical development of drugs for ultra-rare diseases in the US and EU are different. Whereas a comparative PK study with a surrogate efficacy endpoint and a post-approval commitment study to clinically validate the surrogate efficacy endpoint, are required for US licensure, both PK and clinical efficacy data are needed for EU registration at the time of submission. Using historical efficacy data for comparison would be an option however, published data are often not detailed enough in order to make a proper power calculation.

DISCUSSION: Different drug companies are coming to the market with new fibrinogen products. Due to a limited number of affected patients, how could the feasibility of studies is confirmed? This is a difficult situation as the number of bleeding episodes for each patient is not high enough (2-3 mean of 0.2 bleed/year; Peyvandi 2006) and a large number of patients evaluated for a long period of time could be necessary.

Ramin Tehranchi - Congenital FXIII Deficiency, Clinical and Regulatory Challenges

Rare bleeding disorders encompass inherited abnormalities of haemostasis that may present significant difficulties in diagnosis and management. The overall frequency of these disorders in the general population is low, which makes it very difficult and challenging to conduct well-controlled randomised clinical trials. Regulatory requirements which have progressively increased over time have resulted in an increase in both trial size and duration, adding further complexity to trial conduct in these rare populations. Congenital coagulation factor XIII (FXIII) deficiency, as an example of a rare coagulation disorder, is an autosomal recessive and life threatening bleeding disorder with an estimated prevalence of 1 per 2 to 5 million individuals worldwide. A clinical development program has been developed to investigate the efficacy and safety of recombinant FXIII (rFXIII). This presentation describes the clinical and regulatory challenges in a state-of-art rFXIII development for treatment of an ultra-orphan disease.

Debra Bensen-Kennedy - Challenges in clinical development of novel coagulation factors

Ongoing technical advances in biomedical research have afforded the opportunity to engineer novel agents that may provide advantages to current treatment modalities for patients with rare diseases. However, with this opportunity comes a complex set of challenges. Traditional drug development looks to address relatively common disorders occurring in larger populations. When dealing with common diseases, novel agents can be introduced in a stepwise fashion leading to large studies which can adequately account for underlying variability within the study population and detect small treatment effects. This is not possible with rare disease populations, especially those with disorders of coagulation. In addition to the expected challenge of identifying an adequate number of suitable patients for clinical study, development can be further complicated not only by the need for study globalization but also by issues of endpoint clarity, assay variability and regulatory expectations that differ among countries. When working within the rare disease populations, one must remain cognizant of the impact of the development process on these small groups of vulnerable patients, while maintaining expected development standards. This is true because there are no global rules from regulatory agencies: it is necessary a strong collaboration and strive for standardization (endpoint, assays standardization).

Manuela Scarpellini - Ligneous conjunctivitis and plasminogen deficiency: An experience of an orphan drug development for an extremely rare disease

Ligneous Conjunctivitis (LC) is a rare coagulation disorder characterized by Type I Plasminogen deficiency. The prevalence of this disease is in the range of 1.6 per million inhabitants. A specific replacement treatment is not available and topical Fresh Frozen Plasma (FFP) is the only therapeutic alternative for LC. In this context, Kedrion, as a partner of the National Health System in the Plasma Derivatives Self Sufficiency Program, has been asked by the Italian Medicines Agency and the Medical Community to consider the possibility to develop research projects in this field. Upon this specific request, a program for the development and supply of a human Plasminogen (PLG) concentrate for the treatment of LC has started in 2006 and is currently ongoing. The product has been successfully developed as eye drop preparation and preliminary clinical data showed its beneficial effect in clinical use. On August 3rd, 2007 and June 7th, 2010 Kedrion has obtained the Orphan Drug Designation for this indication in Europe and US respectively. In the last year, two non-clinical studies aimed at evaluating single and 28 days repeated dose ocular tolerance have been performed in rabbits. The results did not show any local toxicity effect. These preliminary encouraging findings show that a PLG replacement therapy is a good alternative to FFP in patients affected by LC linked to type I PLG deficiency. A randomized, controlled, double blind Phase II/III study to evaluate Efficacy and Safety of Kedrion human PLG eye drop preparation has been planned and should start next year. A registry could be valuable to identify patients and collect data for clinical studies.

DISCUSSION: The data presented by Kedrion was very interesting and there was discussion regarding whether the request of the European and US Regulatory agencies to ask for a case control study using placebo as a control, was ethical and reasonable. Since no other therapy is currently available for such a disease, then it would not be reasonable to treat a patient with a placebo and the preclinical data could be enough. Moreover, a low number of available patients make such study extremely challenging. Agencies answered that the presentation of product seems convincing. However, in absence of data or few available data for preclinical studies, procedures need to be followed, while in presence of convincing preclinical data clinical trial design could be re-discussed.

Section 2. PROJECTS REPORTS – 1
Chairpersons:  M. Shima (Japan) and A. Srivastava on behalf of J. Oldenburg (Germany)

Claude Negrier - Report of Project on Thrombin Generation (C. Negrier, Y. Dargaud, T. Lecompte, R. Luddington, A. Wolberg, H. Hemker): Standardization of the method

The aim for the Project was to propose a methodology that may be considered to be the recommended way to perform the thrombin generation test so as to bring standardization in studies performed using this method.

The Project recognized that several variations of the method to perform the thrombin generation test exist in the literature with regard to reagents, instruments and interpretation. However, there is a common feeling that the most used so far and the one on which most work has been done is the CAT assay (Hemker et al.). The WP however recommends that a thorough description of the method should be done in any paper on the subject.

Literature published in 2010/2011 has been carefully followed and some lab data and information exchanged. Particularly, further activities to minimise the variability of the assay have been carried out among 5 European centers and the results were published in Thromb Res 2010. The Project recognizes that further activity should be carried out in this direction, and particularly on the use of a reference plasma, the influence of the type of phospholipid vesicles or platelet membrane, as well as and the type and concentration of tissue factor used. It was also felt that the potential impact of temperature should be evaluated and in vitro samples will be soon exchanged among different European (n=4) and USA (n=1) centers. The educational material (DVD) has been updated (2 languages) and should support the TGA activities in different centers worldwide. The availability of the TGA machines has been worked out and most of the participating centers are equipped yet.

In addition to these standardization activities of the CAT assay, the members of the Project felt that the current variability of the assay was good enough to initiate translational research. In this regard, 3 main fields have been thought of:

• Use the common method in 10 centres (5 NA, 5 EU) as a validation exercise for evaluating single clotting factor deficiency (FVIII, FIX, FVII, FXI…) and try to correlate with the bleeding phenotype (if score available) – short term
• Replication exercise in 10 centres (5 NA, 5 EU) on the use of the pilot study done in Lyon, using TGT in surgery carried out in patient s with inhibitors receiving bypassing agents (NovoSeven®, FEIBA®) – short term.

These 2 first exercises should be completed before claiming a complete validation of the assay and a wider availability of the educational material (DVD produced in Lyon) for this purpose.

• Implementation of TGT in prophylaxis (patients with and without inhibitors) – midterm.

Results of monitoring of FEIBA and rFVIIa were presented and it seemed that the latter worked better.

The amount of work needed to standardise this method goes beyond the time dedicated by the FVIII&FIX SSC, hence collaboration with clinical parties is planned.

DISCUSSION: A document providing the details of pre-analytical and analytical aspects requiring standardization will be prepared by the project group before the end of the year and also an educational video on the performance of this test will be made available. This will be published as a report of the SSC subcommittee on FVIII/IX.
Meera Chitlur - Report of TEG/ROTEM Project (B. Sorensen, M. Chitlur, G. Rivard, G. Young, D. Lillicrap, K. Mann, M. Shima, J. Oldenburg): Future strategies

Thromboelastography/Thromboelastometry (TEG) is a global assay used to assess coagulation in a physiologic milieu where all cellular and enzymatic components are preserved. The assay extends beyond the initiation of clotting, and provides information on quantity and quality of clot and fibrinolysis.  Modifications of the assay have been used to assess platelet function and receptor inhibition. The applications for use are wide and growing, extending from hematology and transfusion medicine to cardiology. Today TEG is used to provide information on appropriate product replacement in trauma and surgery. It has been shown to significantly decrease the use of blood products in the peri-operative setting. In the field of hematology, TEG is used to define the phenotypic variation in congenital bleeding disorders (CBD) and tailor replacement therapy with factor products. It remains one of the few assays that can be used to assess the effect of bypassing agents making it extremely useful during surgery and individualizing replacement therapy in CBD patients. Assessing combination therapy with bypassing agents/factor products and antifibrinolytics can only be done with global assays like TEG. One of the areas of investigation has been to predict the risk for bleeding in patients with bleeding disorders or liver disease, providing a means to individualize therapy. With the advancement in medical technology and the use of supportive devices such as external cardiac pumps, monitoring of anticoagulation becomes a major challenge. TEG provides a unique method to evaluate effect of multiple anticoagulant and antiplatelet agents used in combination. It is also being considered for monitoring anticoagulation in patients on extracorporeal life support. TEG has significant potential in assessment of hemostasis but a major drawback remains the lack of standardization.

Results of studies on different disorders monitored by TEG were reported (Glanzmann Thrombastenia, Afibrinogenemia, and FXIII deficiency), TEG was also used to monitor coagulation on ECMO demonstrating an enormous potential for evaluating thrombosis and bleeding.

DISCUSSION: A document describing these results is planned to be published within few months in order to proceed and satisfy the Project mandate.

Midori Shima - Report of Project on Clot wave form analysis (M. Shima, S. Nair, J. Thachil, A. Srivastava): Application to currently available clot detection instruments

Clot waveform analysis is based on the monitoring of transmittance during the routine coagulation assay such as aPTT or PT by automated clot detection instrument.  In addition to qualitative evaluation of the clotting function by waveform pattern, it is possible to assess by various quantitative parameters such as coagulation velocity and acceleration. Although the technique was originally developed by MDA-II (Organon teknica) and this is no more available, many of current automated clot instruments have capability of waveform analysis. We extracted raw data from CS-2000i (Sysmex) and processed the data to derive each parameter. Maximum coagulation velocity, acceleration and deceleration may be good parameters for assessment of global clotting function. Parameters were well correlated with FVIII level in severe cases. Variability was observed in moderate and mild. The next important point is to validate the method on such instrument. Parameters were also reflecting clinical severity. Currently the method is under study on other instruments to finalise result in a manuscript.

DISCUSSION: How specific is the method? The method was working well with all PT and aPTT reagents, but, for example, microparticles were excluded from the tested samples, it still needs to be done.

A document describing the pre-analytical, analytical and post analytical variations should be described and published within few months in order to satisfy the Project mandate. During the year 2012, this group could proceed with a study on a normal population and a group of haemophilic patients, preferably with low levels of FVIII <1% as well as heterogenous clinical manifestations in order to understand the sensitivity of this assay in a low range of FVIII, and to correlate minimal FVIII activity with the clinical severity of patients..

Session 2: 14:00 – 18:00

Section 3. CLINICAL ISSUES (inhibitors, prophylaxis, novel therapies)
Chairpersons: K. Fischer (The Netherland) and C. Hay (United Kingdom)

Donna DiMichele - Considerations for optimal clinical trial design for new clotting factor concentrates

After all discussions during the morning sessions, it is clear that new products for treatment of hemophilia A pose several concerns and new questions, in particular for pre-licensure studies.

The Design of Clinical Trials in Hemophilia Project Group (PG) proposed by Flora Peyvandi and Alok Srivastava and now chaired by Donna DiMichele began its deliberations in February 2011 and has continued its work through a series of 7 teleconferences to date.

The mission of this PG will be to determine the optimal prospective pre-licensure and observational post- licensure trial designs for new clotting factor concentrates for hemophilia on the basis of :1) the harmonized safety and efficacy data required by regulators for registration; 2) the anticipated available study population; and 3) innovative clinical trial design.

This will be accomplished by using consensus definitions (provided to the Group by the Definitions Project); and guidance from regulators in the US and Europe, industry, scientific and methodological experts, as well as clinical investigators. The plan is to initially focus on generating evidence-based recommendations for the conduct of new clotting factor trials in hemophilia A and B and to present these recommendations as they develop to the SSC of the FVII/IX Subcommittee. The PG’s work will be ongoing for a period of at least 2 years and will be further harmonized as appropriate with WFH efforts. After 2 years of activity a manuscript will be prepared and sent as SSC a communication.
Its membership is as follows:
Chair: Donna DiMichele/ Members: Flora Peyvandi, Alok Srivastava, Nisha Jain (FDA); Anneliese Hilger (EMA); Sebastien Lacroix-Desmazes; Frits Rosendaal and John Scott (FDA).

Charles Hay - ITI study: results

The ITI study was a randomised comparison of high and low-dose ITI in good risk subjects. It was stopped on 13/11/09 because of DSMB safety concerns because there were significantly more intercurrent bleeds in the low-dose arm and a recalculation of power suggested that a much larger sample size would be required to prove equivalence. 134 subjects had been recruited at that point. No difference in efficacy could be shown between treatment arms. There was significantly greater bleeding in the low dose arm, largely (85%) limited to the period before the Bethesda titre became negative. Infection had no significant effect on outcome. Of the variables investigated in a logistic regression analysis, only peak historical titre and peak titre on ITI predicted the outcome of ITI.

After the study finished, further data accrual and data cleaning and the very slow process of retrieving samples for central testing and ancillary studies took almost a year. Only then were we in a position to analyse the data. The manuscript of principle results has now been submitted for publication to Blood and subsidiary analyses are in progress. These include pharmaco-economic analysis and modelling and further analysis of the morbidity associated with ITI, focussing on the epidemiology of bleeding and the epidemiology of catheter infection during ITI. Several satellite studies involving study samples are also in progress.

Claude Negrier on behalf of Manuel Carcao - Prophylaxis in patients with inhibitor

Most patients with severe hemophilia will bleed frequently and will develop arthropathy if they are not placed on prophylaxis with factor concentrates. Prophylaxis has consequently become standard of care for non-inhibitor patients with severe hemophilia. Patients who develop high-titre inhibitors have in general not been placed on prophylaxis and instead have continued to be treated on demand and have consequently tended to bleed frequently into joints and develop disabling arthropathy. It should be recognized that treatment of such patients even on demand is very expensive due to the very high cost of bypassing agents.

Until recently there were no prospective or randomized studies on prophylaxis in inhibitor patients. Most of the evidence for prophylaxis in such patients consisted of case reports or small case series. Fortunately in the last few years larger case series have been reported and additionally prospective randomized studies (Konkle et al, 2007; Leissinger et al, 2010) have recently been completed on the use of both bypassing agents for prophylaxis in patients with inhibitors. These studies demonstrate that prophylaxis with bypassing agents in patients with inhibitors appears to be effective in reducing bleeding frequency by about 50-60% in patients who were generally older and have a history of frequent joint bleeding. It should be noted that this is still not as effective as prophylaxis with FVIII/FIX in non-inhibitor patients. The very high cost of prophylaxis with bypassing agents makes it imperative that appropriate studies be undertaken to address the cost-benefits of different bypassing agent prophylaxis regimens in inhibitor patients. But which inhibitor patient should be placed on prophylaxis?

The International Prophylaxis Study group (IPSG) consists of hemophilia experts committed to studying prophylaxis in patients with bleeding disorders. The IPSG was created 10 years ago. Recently it has recognized the increasing importance of, and need for, prophylaxis in inhibitor patients and has established a small group in this area. This expert project consists of experienced hemophilia treaters [M. Carcao (Co-Chair; Canada); C. Negrier (Co-Chair; France); C. Leissinger (USA); E. Santagostino (Italy) and L. Valentino (USA)] whose mission it is to seek new knowledge in this field and disseminate this knowledge globally. In this regard this presentation represents the first presentation of this group at International conferences. This is a not an industry assembled group. It is unbiased and has multinational representation; the main limitation is the lack of funds.

DISCUSSION: The results of the studies show that the rate of bleeding is still significant, and costs are prohibitive. Is not this disappointing? Yes, however, the rate is reduced, even if not at an optimal level.

ON GOING CLINICAL STUDIES ON INHIBITOR DEVELOPMENT:

Pier Mannuccio Mannucci – The SIPPET study

Clinical data stemming from a systematic review that has summarized a few retrospective studies suggest that the choice of the source of FVIII used for replacement therapy (plasma or recombinant DNA technology) might have some influence on the cumulative incidence of inhibitors. A more recent systematic review and meta-analysis of the available data found a two-fold increased inhibitor incidence with the use of recombinant factors, but the difference in favor of plasma-derived FVIII was no longer statistically significant when variables such as study design, study period and frequency of inhibitor testing were included as confounders. To tackle this state of current uncertainty on this issue, the randomized prospective SIPPET trial, undergoing now in 19 countries from 5 continents, is planning to enroll 300 previously untreated or minimally treated patients at risk of developing FVIII inhibitors, in order to establish whether or not plasma-derived FVIII products are less immunogenic that recombinant products. At this time, SIPPET has already enrolled nearly half of the planned number of cases.

Marijke van den Berg - The RODIN study (intensity of treatment and prophylaxis)

H.M. van den Berg, S.C. Gouw, J.G. van der Bom. On behalf of the PedNet and Rodin Study Group: N Clausen, Aarhus; H Platokouki, Athens; M Williams, Birmingham; G Auerswald, Bremen, B Nolan, Dublin; A Thomas, Edinburgh; W Kreuz, Frankfurt; A Molinari, Genova; W Muntean, Graz; A Mäkipernaa, Helsinki; Ch van Geet, Leuven; R Liesner, London; R Ljung, Malmö; H Chambost, Marseille; K Kurnik, Munich; A Rafowicz, Paris; R Pérez Garrido, Seville; P Petrini, Stockholm; S Claeyssens, Toulouse; K Fischer, Utrecht; R Kobelt, Wabern; C Altisent, Barcelona; J Oldenburg, Bonn; E Chalmers, Glasgow; V Jimenez, Madrid; E Santagostino, Milan; G Rivard, Montreal G Kenet, Tel-Hashomer; M Carcao, Toronto; A Cid, Valencia

In the early nineties it has been demonstrated that some plasma products had a higher risk on inhibitor development than other products. From that time onwards many studies collected data on inhibitor formation to correlate with plasma or recombinant products. Since PUP studies are difficult to perform the number of individuals that were included in these studies were too low to investigate properly other risk factors. Data from monozygotic twins have clearly demonstrated that besides genetic risk factors, treatment related risk factors play an even important role. Moreover the advantage of treatment related factors is that they may be changed in an individual patient and as a consequence reduce the inhibitor risk. In the Canal study the effect of prophylaxis as an important factor to reduce the inhibitor risk was clearly stated.

The Rodin study is a joint research effort of 30 haemophilia centers and examines patient-related, treatment-related and environmental risk factors of inhibitor development in previously untreated patients with severe hemophilia A. At the ISTH the first results of the Rodin study will be presented, we preferred for the SCC meeting to give an overview of the patient data both in the Canal study and in the Rodin study. In the Canal study the data were collected up to 50 exposures. Since we could not exclude that some patients developed an inhibitor after 50 exposures, the Rodin study collected data up to 75 exposures. For comparison in this abstract we also made a cut- off for the Rodin study up to 50 exposure days.
 
Full cohorts of consecutive patients were included with a FVIII activity <0.01 IU/mL born between 1990- 2000 for the Canal study and from 2000 – 2010 for the Rodin study. The outcomes were inhibitor development (at least two positive inhibitor titers with a decreased recovery) and high responder inhibitor development (peak titer of at least 5 Bethesda units per mL). We could include n=366 patients with severe hemophilia A for the Canal study and 606 patients for the Rodin study. Data on gene defect were available in both studies for up to 90% of the patients. Also the variability in gene defect was comparable between the 2 studies. The first results on 606 patients show cumulative incidences of all inhibitor and of high responder inhibitor development of 32.0% (95% confidence interval (CI) 28.1-35.9) and 22.2% (CI 18.7-25.8), respectively. In the Rodin study 5 patients developed an inhibitor between 50 and 75 exposure days. Major peak treatment moments at first exposure were associated with a 117% (CI 1.55-3.03) increased inhibitor risk. Patients started earlier on prophylaxis in the Rodin study in comparison with the canal study; however the total incidence of inhibitors did not decrease.

These findings demonstrate that treatment-related risk factors such as prophylaxis and periods of intensive treatment are related to the risk of inhibitor development in patients with severe hemophilia A. Although prophylaxis has an effect to decrease the risk of inhibitor development the incidence of high titre inhibitors was similar between the studies.

Mike Makris - European Haemophilia Safety Surveillance System (EUHASS): update

Adverse event monitoring in persons with inherited bleeding disorders is hampered by low patient numbers. The European Haemophilia Safety Surveillance (EUHASS) was set up as a prospective, multicentre, multinational registry monitoring adverse events.

Since October 1st 2008, acute reactions, transfusion transmitted infections, inhibitors, thromboses, malignancies and deaths in all persons with bleeding disorders were prospectively reported every 3 months by participating European haemophilia centres.

In the first two years data were reported by 64 centres from 27 countries caring for 23,811 patients with inherited bleeding disorders. 12,408 persons had haemophilia A (severe in 5323) and 6968 were treated with concentrate. Rarer bleeding disorder patients were also included eg 346 patients had FX deficiency of whom 44 were treated. 59 different concentrates were used during the surveillance period. A total of 408 events were reported, including 54 acute or allergic reactions of which 4 were anaphylactic; 34/54 reactions occurred within 10min of concentrate administration. Overall there were 95 reports of new inhibitors and 16 of recurrences. No transfusion transmitted infections were reported. 28 thromboses were reported within 30 days of concentrate administration including 5 in FVII deficient patients. Of 83 malignancies 18 were hepatocellular carcinoma and 19 were other gastrointestinal neoplasms. 113 deaths were reported of which 26 were due to bleeding (intracerebral in 17).

Despite the rarity of adverse events to concentrates in persons with rare disorders, the multinational approach allows valuable active monitoring without selection bias and analysis of the safety of multiple products to be rapidly performed. Website: www.euhass.org.

DISCUSSION:

• Is the 2.3 per 1000 patient years inhibitor incidence reported in PTP hemophilic patients comparable with the reported literature? Yes, this is similar to published rates. EUHASS data should be sufficient to identify a problem with a product similar to what occurred in the Netherlands and Belgium in the early 1990s when a product was associated with a much higher rate of inhibitors in PTPs.
• Patients with FVII deficiency are not protected from thrombosis: how can we manage this problem? Since most of the thrombosis cases were associated with a surgery and in some cases even thromoprophylaxis has been performed the use of thromboprophylaxis in FVII deficiency was proposed. Guidelines on the management of surgery in FVII deficiency are required to balance the risk of bleeding and thrombosis in the perioperative period.
• Project is at the final stages, how should the program continue? New applications:  EU does not fund the same project to continue the study. An EU application for a related project that can also cover EUHASS has been made. Moreover, South Africa and Canada are likely to begin collecting prospective safety data using the same protocol and possibly same software this year.

Section 4. PROJECTS REPORTS – 2
Chairpersons: F. Peyvandi (Italy) and L. Valentino (USA)

Paula Bolton-Maggs - Report of Project on definitions in RBDs (F. Peyvandi, D. DiMichele, A. Shapiro, P. Bolton Maggs, C. Lee, C. Negrier A. Tripodi, A. Srivastava): Update

Although there are established definitions for haemophilia A and B (‘mild’, ‘moderate’ and ‘severe’) it is clear that these are not appropriate for other coagulation factor deficiencies.  The task of this Project is to consider whether we can arrive at any consensus for definitions in the other disorders.  The initial approach was a two-pronged retrospective review.

First, a detailed literature search was undertaken (Isabella Garagiola and Andrea Cairo - Milan group) of the English language 1990-March 2011. This sought all reports which included laboratory phenotype, results and clinical details for at least 5 patients. This resulted in 45 papers and 39 review articles.

Second, data analysis from four registries gave additional information about more than 4000 patients. These registries were the European registry (EN-RBD), the UK haemophilia doctors’ organization registry (UKHCDO), the North American database (NARBDR) and the Indian registry held at Vellore.  These four registries did not collect identical datasets.

The analysis overall demonstrated that the RBDs are heterogeneous and complex and a single unifying classification is not possible. Some general conclusions can be drawn: there is a generally poor correlation between factor level and bleeding tendency for both FVII deficiency and FXI deficiency, and some evidence for this also in FV deficiency. There is a reasonably good correlation between bleeding risk and level for FII, FX and FXIII.  FX deficiency and FXIII deficiency are the most serious disorders.

A report summarizing our findings has been circulated to additional experts in the field for comments and there is general agreement that individual factor measurements are insufficient to define the clinical bleeding risk, and that we need to define the role of other global haemostasis measures in the rare disorders. A proposal is put forward shown in the table for definitions:
 
But this is a very preliminary study; it is important to develop better tools for prospective study of these bleeding disorders, and to establish the minimum amount of replacement therapy required under different clinical circumstances.

DISCUSSION: What about genotype? Is it not responsible for the bleeding along with phenotype? Quite interesting, in particular in countries like India or Iran. But in the preliminary analysis this question has not been analysed. In the European and International database, genotype data were collected, but not yet analysed.

Alok Srivastava and Victor Blanchette -  Report of Project on definitions in haemophilia (V. Blanchette, A. Srivastava, M. van den Berg, R. Ljung, M. Soucie, M. Manco-Johnson, N. Key, A. Gringeri)

Evaluation of the safety and efficacy of novel hemostatic agents and of different prophylaxis regimens in individuals with inherited bleeding disorders (focus the hemophilias) requires consistency in definitions of commonly used endpoints.  The mandate of the Project was to perform a critical review of relevant information and to prepare definitions of primary and secondary prophylaxis, type of bleeds (joint, muscle, etc), response to treatment of an acute bleed, target joint and high/low responding inhibitors in the context of long-term factor replacement therapy in persons with hemophilia. Reaching a consensus for some definitions proved to be more challenging, for example, the cut-off between normal and a positive inhibitor level, where a value of ≥ 0.5 Nijmegen-Bethesda Units (NBU) was thought to represent a positive test result. Inhibitors were considered transient if they disappeared within six months of first appearance despite antigenic re-challenge. Another area, still unresolved, is the consensus definition of primary prophylaxis. The current proposal is “regular continuous* treatment started before the onset of osteochondral joint disease, determined by physical examination or imaging studies, and before or immediately following the first clinically evident bleed into an index (ankle, knee or elbow) joint”. Finally, the chosen definition of joint bleed, a commonly used endpoint in clinical trials of new factor concentrates or prophylaxis regimens was “an episode characterized by rapid loss of range of motion as compared to baseline that is associated with some combination of the following: pain or an unusual sensation in the joint, palpable swelling and warmth of the skin over the joint”. Response to treatment should therefore be defined as the resolution of the most prominent feature that defined the bleed. The proposed definitions will be posted on the ISTH website for broad review and comment. Final revisions will then be made to the definitions prior to submission as a report of the SSC subcommittee for consideration for posting on the SSC website and publication in the Journal of Thrombosis and Hemostasis.

*Continuous is defined as the intent of treating for 52 weeks/year and receiving a minimum of 46 treatments/year.

During the presentation the author poses the problem of the definition of “severe” haemophilia A based on laboratory phenotype and wonders whether a fourth class of “moderately severe” needs to be added to the currently used classification. Suggestions were also sought for the content of the draft published on the web regarding results of the Project.

Anthony Hubbard - Report of project on potency labelling of clotting factor concentrates (Hubbard, M. Weinstein, R. Seitz, J. Dodt, T. Lee, A. Srivastava)

A Project has been established to investigate harmonisation of the potency labelling of clotting factor concentrates, focussing on factor VIII and factor IX, which are licensed for different markets. The primary objective is to recommend procedures which will ensure equivalent vial contents for products licensed in different countries and the Project membership includes representatives of licensing authorities from both Europe and North America. New products will challenge the current concept where all product are potency labeling with IU. Strategies leading to a common approach in potency labelling, for both conventional and modified products, will be explored taking into account similar initiatives by other organisations including the European Medicines Agency and the European Pharmacopoeia. A second objective of the Project is to provide recommendations for the potency estimation of clinical samples in the assessment of recovery after infusion of concentrates. In connection with the latter objective a survey has been initiated to review current practice in clinical laboratories. The possibility to use global assays to test potency labeling has been considered, but several cons vs pros need to be evaluated. An algorithm to define potency labeling of new products has been developed. Post infusion testing has the same problems as potency labelling, but remains more confusing: do laboratories use standard to test a sample? A survey was sent to more than 20 labs: only 6 answered.

DISCUSSION: how can we list problems and give priorities to solving them? How can we make comparisons when there is no standard? The most important thing is that information on new product pre-licensure is not available to the community. Manufacturers could be involved in in vitro assays in collaboration with SSC. The project group will also attempt to finalize its recommendation over the next 1 year.

Sven Bjorkman - Report of Project on PK of FVIII/FIX (P. Collins, S. Bjorkman, V. Blanchette, K. Fischer, M. Morfini, E. Tuddenham)

During prophylactic treatment of haemophilia A there is a relationship between exposure (plasma level as function of time) to FVIII and reduction of the risk of bleeding. The achieved exposure after a given dose depends on the pharmacokinetics (PK) of FVIII in the patient. Variance in FVIII PK between patients thus contributes to variance in treatment response and/or FVIII dose requirement. Defining prophylaxis as keeping the plasma level above 1% of normal at all times is an oversimplification. Despite this, PK, in conjunction with evaluation of clinical outcome, is a tool for individualizing dosing of FVIII. With the future introduction of long half-life FVIII analogues, PK considerations may also become increasingly important for rational dosing. Proof of concept has been presented for the use of sparse blood sampling and Bayesian analysis for measuring PK in clinical practice and using this information for dose tailoring, potentially making prophylaxis more cost-effective. The user-friendly TCIWorks (www.tciworks.info) software for Bayesian analysis is freely available for download and has been evaluated for FVIII PK. The Project aims to present a guideline publication that will provide a description of recommended blood sampling schemes as well as of the methodology and application of the Bayesian PK analysis. This publication will also provide necessary input information for practical use of the TCIWorks program.

DISCUSSION: The author has been asked to estimate the time needed for the publication of the paper? The author explained that it is going to need some time since the Advate data need to be accepted for publication and TCIWorks version 2.0 needs to be available and following that they could make their recommendations.

Section 5. STANDARDIZATION ISSUES
Chairpersons: L. Valentino (USA) and J. Oldenburg (Germany)

Steve Kitchen - Monitoring recombinant FVIII concentrates and role of ReFacto AF laboratory standard

Three samples from moderate/severe haemophilia A patients each after treatment with a different FVIII concentrate - ReFacto AF, Kogenate (FS) or Advate - were sent to 57 UK haemophilia centres who determined FVIII in one stage assays (n = 46) or chromogenic assays (n = 10 including 5 different kits). One stage and chromogenic assays were calibrated with a plasma standard or recalibrated with the ReFacto AF lab standard. Taking all results together, irrespective of local assay reagents used, chromogenic assays gave significantly greater results (p<0.0001, 32% difference) in the post Kogenate sample but not in the post ReFacto AF 11% higher by chromogenic assay) or post Advate samples (3% lower by chromogenic). 15 centres used APTT reagents (Synthasil)/deficient plasma/reference plasma from Instrumentation Laboratory in the one stage assay and 20 used all Siemens reagents (Actin FS as APTT reagent). This made a significant difference to results post ReFacto AF (41% higher by IL reagents, p<0.0001) and Advate (39% higher by IL reagents, p<0.0001), but not Kogenate (7% higher by IL, ns). In this study use of the ReFacto AF Lab standard was therefore required for one stage assays to be in agreement with chromogenic when one stage assays were performed using Siemens reagents but not when IL reagents were used. Differences between results obtained with different one stage assay reagents for monitoring Advate have implications for dosing patients, if confirmed. Several different chromogenic assays were used by participating centres and the CV of chromogenic results was high, so more data on different chromogenic and one stage assays to monitor Kogenate are required.

Benny Sorensen - Standardization of low TF and TF+tPA assays for TEG/ROTEM

Establish a recommended method to perform TEG/ROTEM in haemophilia. Variations of the method are reported in literature, hence a review of the literature is highly needed, to outline similarities and differences.

Method based on continuous measurement of physical changes during blood coagulation. Ten papers on 122 patients with haemophilia: TEG/ROTEM can be performed with minute amounts of TF. Dilution of Innovin causes variable results and variation batch constitute a serious challenge to standardization.

Results: Clotting time has limited dose response while maximum velocity has dose dependent response. TEG/ROTEM needs to be developed in the area of hemophilia to evaluate adjunction of therapy.

The Project has established a list of reagents specification for the method and is currently evaluating different batches with internal validation.

Future: collaborate with the recently formed NIH working-group on global assays with the first aim to develop a business plan and establish a group of collaborators

DISCUSSION:

• Due to high cost of reagents, will manufacturers continue to provide reagents? The author is continuously negotiating with them, but collaboration is not always certain.
• Be aware that by using Corn Trypsin Inhibitor, the sample cannot be re-used, so collection needs to be done with a specific procedure. But is it required to collect sample in CTI? Yes, otherwise response could be underestimated.
• Dr. Sorensen committed on behalf of the project group that he will work towards providing a standardization document by the end of the year so that labs around the world could attempt to perform this test will lesser variables.

Sanj Raut - New approaches in FVIII inhibitor measurement and standardization

Previous inhibitor studies have shown high variability in measurement of FVIII Inhibitors between laboratories with CVs ranging from 40-200%. The classical

Bethesda Assay developed by Kasper et al in 1975 and is still the most widely used inhibitor assay.  This assay requires the inhibitor patient’s plasma sample to be incubated with an equal volume of normal pooled plasma, the “Test Mixture”, at 37°C for 2 hours, after which the residual FVIII activity is measured and expressed as a % of the FVIII in a “Control Mixture” (consisting of equal volumes of normal pooled plasma and buffer) which had undergone the same incubation conditions as the “Test Mixture”.  An inhibitor titre is then calculated in Bethesda Units (BU) per ml using the % residual FVIII:C based on the definition of the BU which is that amount of inhibitor that would neutralise 50% of the FVIII:C in normal plasma in a 2 hour period at 37°C.  More recently, Verbruggen et al, had modified the assay by introducing buffering of normal plasma pool and using FVIII-deficient plasma in place of the buffer in the “Control Mixture” to give a better specificity. However, recent inhibitor surveys revealed that the inter-laboratory variability still remained unacceptably high.

Our laboratory questioned the need for FVIII deficient plasma, which was introduced into the assay as a like-for-like diluent for the Reference “Control”.  Ideally we would like to see identical mixtures in both the Test & Reference “Control” mixtures.  However, we hypothesised that by introducing the FVIII-deficient plasma we were actually introducing a variant in the assay and that this variability can be exacerbated by the many different FVIII-deficient plasmas now commercially available.  More importantly, as the Inhibitor titre is based on % of FVIII in the Reference (“Control”), we should be able to replace the FVIII-deficient plasma with a more like-for-like diluent such as Buffered Normal Plasma. The unknown inhibitor titre in this assay (South Mimms Inhibitor Assay - SMIA) would now be expressed relative to 200% FVIII in the Reference “Control” (rather than 100% FVIII previously).  Furthermore, this approach would remove the variant; and reduce a significant step in the inhibitor assay, but more importantly, significantly reduce the cost of an inhibitor assay.

Surrogate inhibitor patient samples were developed by spiking A/FVIII neutralising MAbs & polyclonal antibodies into FVIII-deficient plasma at a range of different concentrations. The above hypothesis was then tested by comparing Nijmegen inhibitor assays with the SMIA test using these samples.  Results showed that, for high titre samples (40 -5 BU/ml), equivocal inhibitor titres could be obtained using the 2 methods. For low titre (1.0 - 0.15 BU/ml) samples, Nijmegen assays were able to detect inhibitor titres down to ~0.6 BU/ml, whilst SMIA tests were able to detect inhibitor titres down to ~0.2 BU/ml.

SMIA tests can produce equivocal results, without the need of FVIII-deficient plasma and is more sensitive to low titres (up to ~0.2 BU/ml) than compared to the Nijmegen assay. SMIA will have a significant assay step removed from the assay, which will significantly reduce its cost. It will be a very simple and a welcome modification to the assay by clinical testing laboratories. It was therefore proposed that a collaborative study be carried out, on behalf of the FVIII/FIX subcommittee, to assess the inter-laboratory variability and sensitivity of this assay, compared to the Nijmegen assay.

Steve Kitchen - Standardisation of assays for rare bleeding disorders: data from UK NEQAS

There continues to be some variability in the detection rate of abnormalities when different PT and APTT reagents are used for initial screening tests. For a sample with FXI of 25 IU/dl 30/ 750 (4%) of centres participating in a UK NEQAS survey reported a normal APTT, whilst a further 80 (11%) considered their results to be borderline. Detection of a 23 IU/dl level of factor VII was better, with 94% of centres reporting abnormal PT results. The variability of factor assays for FII, V, VII, X and XI in 260-290 participating centres was similar to that observed for FVIII and IX assays. In all case this variability was greater when factor levels were lower, as indicated by greater CVs, which were in the range 9 -17% at normal factor levels, and 13 to 26% when factor levels were in the 20-30 IU/dl range. Typically centres obtain PT/APTT reagents, reference plasmas and deficient plasmas to construct assays from the same commercial sources. Comparing use of different reagent sets shows that there are often 10 to 20 % differences between results obtained in factor assays. Finally in one survey of assay design 1/3 of centre use only a single test dilution and had more than double the variability of centres using 2 or 3 dilutions (as indicated by between centre CV).

DISCUSSION: since the data are coming from immunodepleted plasma of patients with FXIII deficiency.  Flora Peyvandi offered collaboration in order to have data on plasma of congenital FXIII deficient patients with very low levels of FXIII. The role of blanking should also be mentioned.

Closing remarks – Flora Peyvandi

In her closing remarks, the chair thanked all the co-chairs, speakers and attendees for their participation and closed the meeting at 17.50 on the 23rd of July, 2011.

The chair asks the attendees to have a look to the recommendation published on the web; all new suggestions will be very much appreciated.