54Th Annual Scientific and Standardization Committee Meeting

54th Annual Scientific and Standardization Committee Meeting

Vienna, Austria

July 2-5, 2008 SCIENTIFIC SUBCOMMITTEE MINUTES

2-5 July 2008, Vienna, Austria

Table of Contents Animal, Cellular, and Molecular Models of Thrombosis and Haemostasis ...... 3 Biorheology ...... 5 Control of Anticoagulation ...... 7 Disseminated Intravascular Coagulation (DIC) ...... 15 Registry of Exogenous Hemostatic Factors ...... 17 Factor VIII and Factor IX ...... 19 Fibrinogen and Factor XIII ...... 30 Fibrinolysis ...... 33 Haemostasis and Malignancy ...... 37 Lupus AntiCoagulant/Phospholipid‐dependent Antibodies ...... 38 Perinatal/Pediatric Hemostasis ...... 41 Plasma Coagulation Inhibitors ...... 44 Plasma Kallikrein‐Kinin System ...... 47 Platelet Immunology ...... 49 Platelet Physiology ...... 54 Predictive Variables and Cardiovascular Disease ...... 56 Vascular Biology ...... 57 Von Willebrand Factor ...... 60 Women’s Health in Thrombosis and Haemostasis ...... 65 Working Group on Coagulation Standards ...... 67 Working Group on Gene Nomenclature ...... 69

Animal, Cellular, and Molecular Models of Thrombosis and Haemostasis

5 July 2008 Vienna, Austria

Chair: Hartmut Weiler, USA Co-Chairs: Shaun Coughlin, USA; Jay L. Degen, USA; Cornelis Kluft, The Netherlands; Nigel Mackman, USA; Tim Nichols, USA; Susan Smyth, USA

The Symposium "Appropriate Use of Animal Models in Preclinical Development of Hemostasis and Thrombosis Therapeutics" was held on July 5, 0800-1130.

Time Subject / Title; Speaker

Animal Models of Thrombosis and Hemostasis for Pharmaceutical Development: Industry´s Perspective Eva-Maria Muchitsch; Baxter AG, Vienna

Dr. Mutchich provided an overview of regulatory requirements and guidelines governing the development and preclinical testing of anticoagulants in preclinical animal models. Discussions highlighted interest to feature this topic in a potential collaborative review to be submitted as a publication of the working group.

Multiyear Safety and Efficacy of AAV-Mediated Gene Transfer For the Hemophilias Timothy Nichols; Univ. North Carolina; Chapel Hill; U.S.A

Dr. Nichols summarized and up-dated outcomes of long-term studies addressing the efficacy and safety of viral gene delivery to correct the bleeding defects in hemophilic dogs.

Evaluation of a Novel Global Hemostatic Agent in Hemophilia A Dogs David Lillicrap; Queens University; Kingston; Australia

Dr Lillicrap reviewed and extended on recently published findings on the efficacy of sulfated polysaccharides isolated from seaweed to ameliorate bleeding in hemophilic dogs.

Development of Transgenic Mice Through Lentiviral Transgenesis For Hemophilia A and B Robert Montgomery; Blood Center of Wisconsin; Milwaukee, U.S.A.

Dr. Montgomery presented data on lentivirus-mediated gene therapy in mouse models of hemophilia A and B. The studies document the feasibility of cell-type selective expression of fVIII and IX with lentiviral vectors, discussed the potential

benefits of this approach with respect to the presence and development of inhibitory antibodies.

Blood flow devices in animal and man as models of hemostasis and thrombosis Kjell Sakariassen; KellSa s.a.s.; Biella, Italy

Dr. K. Sakariassen (Biella, I) described the use of ex vivo flow models based on a shunt-device in translational medicine. The device resembles a stenotic flow chamber for whole blood assays. The chamber produces shear-rates conducive to rapid platelet thrombus formation, and is suitable for analysis of platelet function as well as coagulation.

Arterial Thrombosis and the Role of Thrombin in Atherosclerosis Hugo ten Cate; University of Maastricht; Maastricht, Netherlands

Dr. TenCate presented data from the analysis of mouse models with defects in the protein C pathway and thrombin function, and how these defects modify the expression of atherosclerosis in mice. Discussions focused on the challenge of modeling in rodents the atherothrombotic phenotypes of plaque rupture/stability.

Imaging techniques for small animal in-vivo explorations: Current possibilities and evolutions Antonio Servadio; ANIMASCOPE, Lyon, France

Dr. Servado gave a comprehensive overview of state-of-the-art non-invasive imaging in small laboratory animals. Approaches included MRI, two-photon- microscopy, and ultrasound in combination with selective molecular probes.

Discussion and Planning Hartmut Weiler

The subcommittee will continue to provide a forum for selective topics on the use of animal models in hemostasis research. Drs. Muchitsch, Nichols, Lillicrap, and Weiler will draft a framework for a potential review of regulatory issues pertaining to the use of animal models in hemostasis research, that could serve as a reference for facilitating the establishment of animal protocols complying with regulatory requirements.

Biorheology

4 July 2008 Vienna, Austria

Chair: J. W. M. Heemskerk, The Netherlands Co-Chair: Thomas Diacovo, USA; Marc Hoylaerts, Belgium; Michael King, USA; Gerard B. Nash, UK

DRAFT

The Biorheology session was an intensive meeting with 11 presentations divided into 2 parts, corresponding to two working parties and two special projects. The session was held at a rather distant location, it attracted only 50 participants.

Introduction Chairman Dr. J. Heemskerk presented a short overview of the working parties and special projects of the subcommittee, as also reflected in the program of this year’s session.

Part 1. Assessment of flow-determined molecular processes in thrombosis and hemostasis (chair: JJ. Zwaginga, T. Diacovo, M. King)

Dr. M. King (Rochester, USA) discussed how to determine the formation rate of single adhesion bonds ( the other half of the story). The presentation focused on advances in mathematical modeling of platelet tethering and adhesion to VWF (small and large multimers). He demonstrated how to evaluate 2-dimensional rates of bond formation. Also a technique was presented to analyze the kinetics of bond formation using a cell attached micromanipulator system. Dr. T. Diacovo (New York, USA) gave a presentation entitled: Role of the von Willebrand factor A1 domain in flow-induced thrombus formation: therapeutic potential. Mutant VWF A1 domains were engineered on the basis of expected changes in their binding to GPIb. Coating of these domains to microspheres allowed to study the interaction with platelets. The in vivo relevance of the modified A1 domains was furthermore shown in mouse models. Injection of human platelets, whether or not pretreated with antiplatelet drugs, allowed determination of the significance and the therapeutic potential of the VWF A1-GPIb bond formation. Small changes in amino acid composition appear to lead to considerable effects on thrombus formation in vivo.

Dr. J.J. Zwaginga (Leiden, NL) spoke on blood flow assays in the characterization of von Willebrand disease: need for a plasma biobank. He discussed the recently published position paper of the Subcommittee on Biorheology, stating which and how flow assays could best contribute to explain remarkable phenotypes of VWD patients (bleeding vs. non-bleeding). This paper describes technical and analytical issues of commercial and non-commercial flow chambers, which are important for proper testing of blood samples. The next step however is to test VWD plasma samples (reconstituted with donor platelets and erythrocytes) in various flow chamber systems in the form of a multi-centre study. The possible advantage of applying flow vs. ristocetin should appear from such preclinical testing. The working party proposes to generate a biobank of VWD plasma samples for testing in flow assays. The framework for this should be a working party together with the SSC Subcommittee on VWF. Subsequently, Dr. G. Nash (Birmingham, UK) gave a presentation entitled: Models for studying the responses of endothelial cells to shear stress. He underlined the importance of these models, given the regulatory role of endothelial cells in thrombosis. One facet is the readout of flow effects on gene expression; KLF2 can be used here as a standard marker.

On behalf of Dr. R. Farndale (Cambridge, UK), Dr. J. Heemskerk presented data showing the advantages of using collagen-derived peptides as a thrombogenic surface in flow chamber studies in comparison to native collagens. Clear disadvantages of commercial and non-commercial collagen preparations are the lack of knowledge of their molecular composition and the huge variation in size of the collagen fibers. This can explain part of the current variability in measurements of thrombus formation. Dr. J. Heemskerk further presented data of the working party on standardization of thrombogenic surfaces in flow. Promising effort has been made in the use of collagen-derived peptides as well in the application of coagulation in flow-based assays.

Part 2. Determinants of thrombus formation under flow in vitro, ex vivo and in vivo (chair: M. Hoylaerts, G. Nash, J Heemskerk)

Dr. B. Furie (Boston, USA) presented data on novel findings and novel technology to study thrombus formation in vivo. He described key characteristics of the in vivo model of laser-induced injury. Current techniques also allow to study activation responses in endothelial cells close to the site of injury. Dr. A. Reininger (Munich, G) gave a presentation, entitled: does thrombus formation under flow adequately measure platelet and clotting activation? He showed that a multitude of (new) factors/processes has an important influence on the size and structure of thrombi under high-shear conditions: VWF mutations, stretching of VWF, platelet tether formation, fibrin production by aggregated platelets and the microenvironment in aggregates.

Dr. Ph. De Groot (Utrecht, NL) gave a presentation on multiple substrates in flow-induced platelet adhesion. He reviewed the evidence for interactions of the GPIb complex with multiple plasma factors, including coagulation factors II, VII, IX, X, XI, XII, HK and PC. In many of these cases, the interaction requires the presence of a second adhesive platelet receptor, e.g. LRP8. Dr. K. Sakariassen (Biella, I) described the use of ex vivo flow models in translational medicine. The use of a stenotic flow chamber, with high wall-shear rates, leads to a remarkable increase in thrombus size. This type of flow chamber also incorporates coagulation.

The chairman thanked speakers and audience for presentations and extensive discussions. The meeting was then closed.

Submitted by J.W.M. Heemskerk

Control of Anticoagulation

4-5 July 2008 Vienna, Austria

Chairman: Sam Schulman, Canada Co-Chairs: W. Ageno, Italy; T. Baglin, UK; J. Harenberg, Germany; C. Kearon, Canada; A. Lubetski, Israel; J. Olson, USA; G. Palareti, Italy; A.M.H.P. van den Besselaar, The Netherlands

4 July 2008

Chairmen: S. Schulman and T. Baglin

S. Schulman opened the meeting of the committee and briefed on the activities over the past year. As AMHP van den Besselaar was unable to attend the SSC meeting his activities are accounted for here:

1. Replacement of the International Standard (IS) for thromboplastin, human, plain. Two candidate materials have been obtained. These have been freeze-dried in glass sealed ampoules. Preliminary tests indicate that the ISI is in the required range. In the next months, a collaborative study by 20 international centers will be carried out for the establishment of the ISI values for these candidates. Results will be presented at the next meeting in 2009. 2. Revision of statistical procedures in ISI calibration. For primary ISI calibration, selection of fresh patients’samples within the therapeutic range is required. A revised selection procedure has been proposed based on the mean INR for each sample from two reagent systems (i.e. INR from reference system and INR from preliminary calibration of new system). For the detection of outlier samples (deviation > 3 SD) in ISI calibration, the current practice can be maintained. If the ISI model used for the calibration of a given thromboplastin PT system leads to clinically important bias, it is recommended to use a modified model which avoids the bias. A draft guideline paper has been prepared and will be submitted to the SSC. 3. Performance requirements of Point-of-Care INR monitors. The analytical performance requirements of POC monitors should be defined. The imprecision of the INR determined with a POC monitor should not be greater than a maximum value which is to be derived from the “biological variation” of the INR. Studies have been started to determine the biological variation of the INR in selected patient groups. Results will be presented at the next meeting in 2009.

Minisymposium. Towards a recommendation for standardization of platelet thrombography Introduction. Trevor Baglin

Preanalytical variables. Thomas Lecompte

Analytical variables. Thomas Siegemund

Interpretation of platelet dependent thrombography. Martin Besser

Open discussion and summary. Trevor Baglin, Nigel Key

Conclusions:

Several of the SSC subcommittees currently have an active interest in the measurement of thrombin generation. Standardisation is required if the full potential clinical utility of this methodology is to be realised. This is particularly so with platelet-dependent thrombography where there are additional pre-analytical and analytical variables to those affecting measurement of thrombin generation in platelet poor plasma. The interpretation of thrombin generation in the presence of platelets also requires consideration of method-specific issues in addition to those relevant to the interpretation of thrombin generation generally.

The purpose of a recommendation from the SSC is two-fold. First it will provide a template of relevant variables that have to be considered in planning and development and for which details that will have to be provided when presenting and publishing work. Second, it will provide a framework for standardisation of these variables where appropriate. In this respect it is not intended that the recommendations for standardisation are didactic but rather that they be used to ensure all issues are considered and dealt with appropriately and with clarity.

The recommendation will therefore provide guidance in relation to pre-analytical and analytical variables and the interpretation of measurement of platelet-dependent thrombin generation. For each variable a recommendation will be made as to the ‘standard method’ with can be used with discretion. This will be based on evidence where such evidence exists and otherwise will be based on established principles of good laboratory practice, simplicity and pragmatism.

Pre-analytical variables will include sampling, specimen tubes, anticoagulant and buffering, control of contact factor activation, timing of samples (fasting, diurnal variation, etc), timing of analysis (delay).

Analytical variables will include tissue factor (source, concentration), platelet count & size, standardisation of platelet count when investigating plasma factors and standardisation of plasma factors when investigating platelets.

Interpretation issues will include reference to platelet count, plasma (PPP) comparator, imprecision (inter and intra-assay CV), normal range, biological variation, and the need for interpretation with reference to the biological context of the problem.

A draft recommendation will be produced by a writing group and be sent to two reviewers in the Autumn before submission for consideration by the SSC. The proposed writing group will be Trevor Baglin, Martin Besser, Yesim Dargaud, Nigel Key, Thomas Lecompte, Alan Nurden and Sirak Petros.

Guidelines on computer-assisted dosage programs for vitamin K antagonist therapy. Leon Poller on behalf of the Advisory Group

Basis for the recommendation is derived from a large study with clinical endpoints, recently published in JTH. The EAA study recruited 18617 patients randomized to manual or computer dosing (Dawn or Parma program) (Italy, UK, Slovenia). Clinical events 6.0 vs 5.5 per 100 patient-years, statistically signif only for VTE (9.1 vs 6.1). It would be very difficult to repeat the study and compare different computer programs. Mean % INR in therapeutic range is used most often for quality assessment. A 6-month follow-up should be included with up to 50 patients at 10 centers giving 500 patient-years. How many new patients should be in the evaluation – 75%? Almost all patients should be targeted at 2.0-3.0 (majority) and 2.5-3.5. In the EAA study the % TIR for one program + reference) was 59.7% (58.5-61.9%). Then 10 centers were selected and evaluated separately for first 6 months of therapy ->60.4% TIR (55.7-65.0%). For the second program =test program ->63.9%. Leon Poller discussed inclusion of patients with <10 weeks treatment, if >6 week interval. A suggestion was to perhaps evaluate intial and chronic patients separately. TIR within limits of EAA is important but also failure-to- dose rate 14.3 – 19.7%.

A draft recommendation for evaluation of computer programs has been sent out and commented on by the Advisory Group. Further revision will be made based on the discussions here at the SSC and a manuscript is anticipated in the very near future.

Chairmen: S. Schulman and J. Harenberg

Minisymposium. Generic low molecular weight heparins – problems, current status, requirements to be fulfilled and further actions J.Harenberg, J. Fareed, and others

Are LMWHs different based on results from clinical studies? Michel Samama

Two LMWHs have been compared head-to-head tinza 4500 IU vs enox 40 mg in 440 patients showed no difference although enoxaparin had higher antiXa levels but tinzaparin had higher antiIIa levels. Secondly, bemiparin 3500 IU vs enoxa 40 mg, again in orthopedic surgery without a difference. Nadroparin versus enoxa in 8000 patients found that 30% less bleeding complications with enoxaparin in orthopedic patients. Dalteparin seems to have lower efficacy and better safety than others. In a Turkish study dalteparin 120 IU/kg bid and enoxa 1 mg/kg bid were equally safe and effective in ACS, although enoxa delayed the time to MI. Tinzaparin has better PK in patients with renal failure. It is still uncertain what the clinical importance is of the biochemical and preclinical differences.

Biosimilar LMWHs – how do we define similarity? Elaine Gray

The differences between LMW heparin and unfractionated heparin and the properties of various LMW heparin products were described. The mean molecular weight and anti-Xa/anti-IIa ratio differ among the products, but there is some flexibility in the specifications for each individual product as defined in the relevant EP monographs. New versions of the existing products are now being produced as “biosimilars” – it is unclear whether these should have exactly the same specifications as the existing EP monographs for the parent compounds, or whether there is a need for additional characterisation. An EMEA Guideline on biosimilar LMW heparins is currently out for public consultation. The general requirements for LMWHs are: Average MW <8000, 60% of molecules <8000, anti-Xa/anti-IIa >1.5, similar NMR spectra. However, enoxa and nadroparin look different on NMR. Tinzaparin has >20% above MW 8000. Should we ask for polysaccharide or end group profiling?

Contaminants in heparins and LMWHs – clinical implications. Jeanine Walenga on behalf of Jawed Fareed

The number of deaths of patients receiving heparin reported to FDA, during the period January 2007 through May 31, 2008 stands at 246, with 149 attributable to one or more allergic/hypotensive symptoms. Contaminated heparin containing oversulphated chondroitin sulphate was linked to the observed clinical adverse events. After the February 28th, 2008 recall and the subsequent monitoring by FDA, it was reported that the heparin associated deaths had returned to baseline (Kishimoto et al, NEJM, 358;23, 2008), but thereafter a total of 30 reported deaths with 17 linked to one or more allegeric/hypotensive symptoms, and other factors such as immunogenic responses may be contributory. Heparin is a very complex drug. While contact system activation may contribute to the reported pathogenesis, it is unlikely to be the sole cause of reported deaths in patients treated with heparin. Additional studies in animal models and retrospective analysis of biologic fluids and available autopsy data on patients maybe helpful in the understanding of these adverse events and deaths (Hoppensteadt et al., Clin Appl Thromb Hemost, 2008;14; 261).

As an initial step, the chemical and biological profiles of the contaminant in recalled unfractionated heparins (UFH), four recalled UFH preparations (3 finished products and 1 powder) were investigated. To obtain the contaminant, each material was treated by exhaustive depolymerization with nitrous acid and heparinase 1 to remove heparin followed by ethanolic precipitation and anion exchange chromatography. The amount of non- digested material ranged 10-30%, most of which was characterized to be hypersulfated chondroitin sulfate (HSCS) by proton and 13C NMR spectroscopy. The molecular weight profile exhibited a wider dispersity index in comparison to contaminant-free UFH with oligosaccharides ranging from 5-30 kDa (average 16.8 kDa). In addition, a well-characterized porcine cartilage HSCS preparation with average molecular weight of 17.2 kDa was used as a reference material. While varying degrees of dermatan sulfate (high molecular weight) and minor impurities were detected, the HSCS appeared to be the major contaminant in these preparations. To investigate the biological profile of the isolated contaminant, it was subjected to chondroitinase A, B, and C and high potency heparinase 1 (2.5 U/ml) depolymerization. The material was resistant to the action of these enzymes. The contaminant was further profiled in routinely used anticoagulant and anti-protease assays. In the USP assay it exhibited a potency of 26.8 U/mg. It also produced a concentration dependent anticoagulant effect in the whole blood celite activated clotting time (ACT) and saline ACT tests but was weaker than heparin.

In the PT assay (extrinsic coagulation system) the contaminant only exhibited very weak activity and did not affect the INR up to a 50 µg/ml concentration. However, in the global anticoagulant assays such as the aPTT (intrinsic coagulation system) and Heptest, in comparison to UFH, the contaminant produced varying degrees of concentration dependent anticoagulant activity (10-40 U/mg). In the amidolytic anti-thrombin assay it produced a concentration dependent inhibition of thrombin in citrated plasma (~ 25 U/mg). This contaminant did not exhibit any inhibition of FXa in the same systems. In antithrombin (AT) depleted plasma, while the anticoagulant and amidolytic activities of UFH were considerably reduced, the contaminant exhibited measurable concentration dependent effects indicating a non-AT dependence. In HCII depleted plasma the contaminant lost sizeable anticoagulant and anti-thrombin effects. The dermatan cofactor activity of pre- and post-heparinase digested contaminant was not different, whereas UFH showed a considerable decrease. The contaminant was readily neutralizable by protamine sulfate, polybrene, and PF4 in a similar fashion as UFH. The contaminant mediated contact factor activation as measured by the generation of kallikrein and bradykinin, was concentration dependent in plasma and whole blood. UFH also showed this activity in both systems. Significant differences in contact activation by UFH and the contaminant were noted between citrated and hirudinized whole blood. The contaminant also produced HIT mediated antibody activation of platelets; however, it had a faster onset of action and longer lasting time course of platelet aggregation than UFH. In the 14C-Serotonin Release Assay (SRA) the contaminant produced a strong release of serotonin which sustained at high concentrations and did not follow the parabolic response usually observed with UFH. Studies of the contaminant mixed with UFH in proportions of 3, 6, 12, 25, 50% (amount of contaminant) in plasma and whole blood revealed a non-additive assay dependent synergistic effect of the contaminant on the anticoagulant and anti-thrombin activities. At a 25% level, the contaminant produced a marked increase of the anticoagulant activity of the mixture mimicking the pharmacopoeial potency of ~ 150 U/mg; however, this increase was dependent on different contaminant preparations (different batches). Similar augmentation of the effects of UFH were noted in the thrombin and FXa generation tests as measured by amidolytic methods and measurements of such thrombin generation markers as FPA, TAT, and F1.2. Preliminary studies show that the contaminant may also exhibit direct anti-protease effects by complexing with FVIIa and the prothrombinase complex. To study the in vivo effect of the contaminant, the effect of contaminated UFH and a potency equivalent contaminant-free preparation were studied in animal models of bleeding and thrombosis. In comparison to the contaminant-free UFH at an identical dosage of 200-800 U/kg, the contaminated UFH produced a marked increase in bleeding. Similarly, the antithrombotic effect in terms of ED50 was markedly stronger with the contaminated preparation. Blood pressure measurements provided variable effects in which both the contaminated and contaminant-free preparations exhibited a hypotensive response in some rats. The contaminated heparin exhibited stronger and longer lasting anticoagulant effects in primates and produced a stronger release of TFPI. Similar studies carried out on two hemi-synthetic HSCS preparations mixed with non- contaminated UFH provided comparable results. Since the contaminated batches of UFH also contain variable amounts of dermatan sulfate, additional studies are in progress at this time on the pharmacologic profile of the contaminated UFH, isolated contaminant, hemi-synthetic HSCS, hyper-sulfated dermatan sulfate, and their precursors. Furthermore, additional investigations on the contaminant isolated from commercially available/branded and generic LMWHs are in progress at this time with particular reference to the molecular profile of the contaminant, its subcutaneous PK/PD profile, and immunogenic potential. Until the availability of such data and additional information on the cause of adverse reactions and deaths in patients treated with recalled heparins is available, the true cause of these events remains unknown.

Generic low molecular weight heparins - assumed requirements of EMEA or FDA. Wolfram Raake

For some of the low molecular weight heparins (LMWHs) the patents have expired and the often raised question is: Why are generic biosimilar medicinal products, especially LMWHs, not under development and cannot be marketed. The answer is: We are missing valid guidelines! The EMEA has established a working group to create a concept paper for similar biological medicinal products containing LMWHs. A draft proposal was published in April 2008 in which detailed requirements for the preclinical and clinical development for biosimilar medicinal products are summarized. In the "Pathway for Biosimilars Act, HR 529, 13 March 2008" the FDA has introduced its ideas for the development of Biosimilars which are basically comparable to the requirements of the EMEA. The research-based pharmaceutical industry has broadly welcomed the bill, but the generics industry is unhappy with it.

EMEA has not insisited on safety pharmacology, reproductive toxicology, mutagenicity or carcinogenicity studies. Clinically, EMEA asks for pharmacodynamic studies with the original substance as comparator in a phase I study. Furthermore equivalent efficacy and safety as the reference in a phase III high risk population

10 such as orthopedic surgery. Extrapolation to other population is allowed. Evaluation of effect on liver, platelets etc also required.

FDA requests analytical, animal PD studies and clinical studies including immunological data.

Requirements for the development of generic LMWHs – current status and future actions. Job Harenberg on behalf of the ad hoc working group of the SSC on Anticoagulation

Job Harenberg presented a draft on recommendations regarding biochemical, pre-clinical and clinical characteristics and requirements for the generic LMWHs that have been discussed at several meetings of the Working Party during the past year. One more meeting is planned for September 2008 and should result in a written statement that can be presented to EMEA as the ISTH SSC opinion.

A new Working Party needs to be formed as specified below: 'Standardisation of methods to determine new factor Xa inhibitors'.

Participants as of July 4th, 2008: J. Harenberg, M. Samama, E. Gray, F. Misselwitz, A. Perzborn. Up to 10 laboratories will be finally participating. Starting date will be: October 2008.

About 6 to 8 anticoagulant assays and 2 to 4 factor Xa inhibitors will be investigated. Reporting dates at the SSC meetings. The expected end date for the final report and manuscript will be Oct. 2010.

Aim: One first line assay, one or two second line assays to determine individually the new factor Xa inhibitor. Sam Schulman will ask the SSC Business Meeting for approval.

July 5, 2008

Chairmen: C. Kearon and W. Ageno

Minisymposium: Harmonization of assessment tool for post-thrombotic syndrome

Introduction of session: Clive Kearon

Post-thrombotic syndrome: Epidemiology, natural history and quality of life. Susan Kahn

Measurement of PTS and evidence for validity of classification systems: a move towards harmonization. Susan Kahn

To date, treatment of established post-thrombotic syndrome (PTS) is unsatisfactory. An important barrier to moving PTS research forward has been a lack of harmonization of the definition and rating of severity of PTS. While there is no objective test to diagnose PTS, a number of clinical scales have been proposed, some specifically for PTS (e.g. Ginsberg; Villalta) and others for chronic venous disease generally (e.g. CEAP, Widmer, VCSS). While each scale has merit, the Villalta scale is proposed as a standard for the harmonization of the diagnosis and rating of severity of PTS in clinical trials and cohort studies of patients with DVT and PTS. The basis for this proposal is that (1) the Villalta scale has established validity, inter-rater reliability, responsiveness to clinical change and acceptability (i.e. ease of use); (2) it considers both subjective symptoms and physical signs; (3) it has been used by various investigative groups internationally to diagnose and rate the severity of PTS in a number of published comparative trials and longitudinal cohort studies; and (4) it can be readily used as a continuous, binary or categorical measure of PTS.

Role of the CEAP classification. Hugo Partsch

The CEAP classification is a worldwide used instrument to describe chronic venous disorders based on their clinical picture (C), etiology (E), anatomic sites of venous involvement (A) and pathophysiology (P). It may serve as a systematic guide in the clinical investigation of patients as an orderly documentation system. An additional severity scoring system is based on three elements: the number of anatomic segments affected, grading of symptoms and signs and disability.

For follow-up studies after DVT CEAP can be used to describe the actual status at any time of re-investigation, preferably also including the starting point at the stage of acute DVT.

Discussion

There appeared to be general agreement for recommending the use of Villalta Score for future studies on PTS, possibly with some additional specifications or suggestions for objectification of the assessment of symptoms, such as with visual analogue tools (since the original publication of the scoring system only is an abstract).

Recent and ongoing trials to prevent or treat post-thrombotic syndrome. Suresh Vedantham

Adjunctive measures to prevent or treat the post-thrombotic syndrome have been the subject of increasing research focus during the past years. Suresh Vedantham summarized recent and ongoing trials that are evaluating endovascular thrombolytic therapies (ATTRACT and CAVENT), elastic compression stocking therapy (Aschwanden trial and SOX), a venous return assist device (VENOPTS), and/or exercise training and attention control (EXPO) for the prevention and treatment of PTS.

Discussion

Acceptable rate of recurrence - after discontinuation of treatment of 1st VTE Discussant 1: Gualtiero Palareti, Alfonso Iorio

An indefinite anticoagulation is recommended in all patients with a previous unprovoked VTE. Some criteria (negative D-dimer after anticoagulation is stopped, complete resolution of thrombus, others?), allow to identify subpopulations at lower risk. Which is the rate of recurrences that can be accepted if we stop anticoagulation in the these patients? We propose that the rate of recurrences occurring in patients with secondary VTE, in whom anticoagulation is recommended for only 3 months, can be acceptable as target limit. Such a rate is, however, not well defined. Dr Alfonso Iorio (University of Perugia) has reviewed the studies reporting on recurrences in provoked VTE and calculated mean rates of 4.7% (95% CI 3.9-5.9) and 7.5% (95% CI 4.2-10.1) after 1 and 2 years, respectively. We suggest these limits be accepted as target of intervention studies assessing procedures to stop anticoagulation in low-risk subpopulations of patients with a first unprovoked VTE.

Discussant 2: Clive Kearon

Trials in which, after an initial period during which all patients receive the same anticoagulant therapy, patients are randomized to either continue or stop therapy are the most rigorous to assess the benefits and risks of indefinite anticoagulant therapy. Using recurrent VTE as the primary efficacy outcome and major bleeding as the primary safety outcome, recent randomized trials have demonstrated benefit to indefinite anticoagulant therapy after a first episode of unprovoked proximal DVT or PE compared with stopping therapy at 3 or 6 months. Comparisons of intermediate durations of anticoagulant therapy, both within and between studies, suggest that the same high rate of recurrent VTE is observed if anticoagulant therapy is stopped after 3, 6 or 12 months in patients with a fist unprovoked VTE (i.e., about 10% in the first year and 30% within 5 years). Because indefinite anticoagulant therapy is burdensome, many physicians and patients are opposed to indefinite anticoagulant therapy after a first unprovoked VTE even though indefinite therapy has been shown to be of benefit in randomized trials. In order for indefinite therapy to be indicated, they argue that the benefits of indefinite therapy need to outweigh its inconvenience and costs as well as its associated risks of bleeding. Consequently, if the risk of recurrent VTE after stopping anticoagulant therapy is low enough, even if indefinite anticoagulant therapy would eliminate this risk without causing much bleeding, indefinite therapy would not be indicated. VTE that is provoked by a minor reversible risk factor such as a medical illness (as opposed to recent surgery) is associated with about half the risk of recurrence that occurs after an unprovoked VTE (i.e.,

12 about 5% in the first year and 15% within 5 years). There is widespread agreement that this risk of recurrent VTE is not high enough to justify indefinite anticoagulant therapy.

In order to be consistent with current clinical practice and the preferences of most patients and physicians, Dr. Kearon proposed that two recurrent VTE rate criteria need to be satisfied for a study to identify an acceptable rate of recurrent VTE after stopping anticoagulant therapy. First, the observed rate of recurrence should be similar to, or lower than, that which occurs in patients with VTE provoked by a minor reversible risk factor (i.e., about 5% in the first year and 15% within 5 years). Second, the 95% confidence interval around this rate of recurrence should exclude a recurrence rate as high as that which occurs after an unprovoked proximal DVT or PE (i.e., about 10% in the first year and 30% within 5 years).

Chairmen: G Palareti and A. Lubetsky

Discussion on Recommendation for Definition of Major Bleeding in Surgical Studies Sam Schulman: A review of the inconsistencies between definitions of major bleeding in surgical studies during the past decade was presented. A previous recommendation by this Subcommittee on the Definition of Major Bleeding in Non-Surgical Studies was taken by the SSC in 2004, published in JTH in 2005 and “quietly” adopted by EMEA in 2006. However, EMEA has recently also published a guideline for a corresponding definition in surgical studies, that partly refers to the above-mentioned SSC recommendation for non-surgical studies and partly suggests other criteria but leaves considerable ambiguity. It is therefore crucial to form a clear recommendation for surgical studies as well. Suggested criteria for further discussion are:

•Fatal bleeding

•Critical organ (probably not counting intra-articular bleeding for joint surgery)

•Requiring reoperation

•Transfusion of >X U whole blood or red cells (5U for example, to demonstrate unexpected major blood loss) and unexpected bleeding for the circumstances (e.g. may be acceptable in revision hip arthroplasty)

To be discussed further: Extrasurgical bleeding causing a fall in hemoglobin level of 20 g/L or more, or leading to transfusion of two or more units of whole blood or red cells.

And separately:

•Blood loss in wound drains, suctions and cell savers

•Transfusions of red cells and whole blood

•Hemoglobin change

The timing of the reporting period has also differed. On one hand, including only the period on study drug removes some noise from the surgery in case study drugs are started only postoperatively. On the other hand, comparisons across study programs and different drugs and regimens would be better understood by showing the totality. This may also be in the interest of the surgical community to understand the expected complication rate and blood loss of each regimen. Discussions have been held with orthopedic surgeons (Bengt Eriksson, Swedemn, Andreas Kurth, Germany and William Fisher, Canada).

Andreas Kurth gave last minute apology for not presenting, due to inability to travel to Vienna this weekend.

General discussion on the definition: There were suggestions to separate the perioperative period (e.g. to 24 h after end of surgery) from the postoperative period and to discuss with a trauma surgeon regarding their concept of massive/unexpected bleeding.

Work will continue during the next 1-2 months to finalize a recommendation.

A new generation of Prothrombin Time method. Juha Horsti

Oral anticoagulant therapy calls for continuous control by prothrombin time test, as the therapeutic range in INR units is very narrow. Warfarin (or coumarin) inhibits the synthesis of K-vitamin depended coagulation factors F II, FVII, FIX, FX in the liver, but at the same time blocked inactive coagulation factors are formed. Liver secretes active and inactive coagulation factors to the plasma. Quick or Owren PT methods measure the sum of inhibition and active coagulation factors in the patient plasma.The new generation PT method measures active coagulation factors and inhibition separately. The new PT method develops anticoagulation therapy based on active coagulation factors in vivo and improves INR result harmonization for Quick and Owren PT reagents.

In vitro effects of direct thrombin inhibitors on fibrin gel permeability. Shu He

The aim was to create a method for examining the effects of thrombin-/Xa-inhibitors (argatroban, bivalirudin, lepirudin, danaparoid) on the fibrin network porosity for the assessment if procoagulants (FVIII) can reverse impairment of hemostasis induced by these inhibitors. The method was based on a modified flow measurement and 3D-micoscopy to determine fibrin network porosity in a normal plasma sample spiked with one of the inhibitors. Argatroban, bivlirudin and danaparoid were similarly potent to inhibit thrombin or Xa and the consequent coagulation, leading to increased fibrin network porosity in a highly dose-dependent way. Although the lack of influenc on fibrin gel porosity by lepirudin at the “plasma-like” levels is in disagreement with the antithrombotic effect of the drug, the extremely sharp dose-response at concentrations above the therapeutic range reflects the clinical experiences of high risk of bleeding from over-dosing lepirudin. In this assay system rFVIII contributed to reversal of the impairment induced by danaparoid and to some extent by argatroban and bivalirudin. This may support the use of rFVIII for treatment of bleeding complications.

Update on International Registry on Recurrent Venous Thromboembolism in Patients with Cancer. Sam Schulman

This registry started 2 years ago, slow recruitment with only 47 cases from 9 sites in spite of reperesentation on website, information spread at meetings, and also by representatives from sponsoring (unrestricted educational grant to ISTH from Leo) pharmaceutical company.

Update on Registry on Splanchnic Vein Thrombosis. Walter Ageno, Francesco Dentali

An international prospective registry on patients with visceral vein thrombosis has been developed. The aim of the registry is to improve the knowledge on this important disorder. Information on most common clinical presentations, most used diagnostic approaches, most common risk factors, therapeutic approaches, and on the two year history of splanchnic vein thrombosis are collected on a web site database. Website address is: http://svt.helloweb.eu Access to CRF will be permitted to each center upon reception of a specific password. Password will be given after contacting the following address: [email protected] The study has been proposed to all ISTH members and is promoted at several national meetings of local societies on thrombosis and haemostasis. We plan to enrol a minimum of 500 patients, including all sites of thrombosis.

Submitted by S. Schulman

Disseminated Intravascular Coagulation (DIC)

3 July 2008 Vienna, Austria

Chair: Cheng-Hock Toh, UK Co-Chairs: Nigel S. Key, USA; Jorn Dalsgaard Nielsen, Denmark; Kenji Okajima, Japan; Hideo Wada, Japan

DRAFT

DIC Report Summary

Theme-Identifying DIC in addressing global health challenges

Standardisation Issues

Geraldine Lavigne presented on her experience of how inclusion of simple coagulation tests had added value to the prognostication of patients admitted to the intensive care unit. A multicentre study was now underway to assess its utilisation prospectively.

Jorn Nielsen reported on the use of the DIC scoring system in recruitment to the ATryn Phase II Clinical Trial. Greater precision was needed from this SSC in defining PT and Ddimer cutoff values.

Moritoki Egi recounted the Okayama validation experience of the non overt DIC score. He found that there was added value in including antithrombin estimations for earlier identification of the process.

Frederic Sobas described the usefulness of the aPTT waveform in identifying patients with sepsis and haemostatic dysfunction. His experience in Lyon is the same as that of other groups in confirming the usefulness of this tool in the critical care setting. He highlighted the limitation of this technology to one analyser only.

Education

With particular emphasis to the relevance of this SSC to healthcare in the under resourced setting, J O Donnell and Christopher Moxon highlighted the relevance of coagulation pathways in malaria. Particular focus was made on the roles of VWF, tissue factor and thrombomodulin. Both presentations were well discussed by attendees to the session.

Collaborations

Armando Tripodi on behalf of the SSC on Control of Anticoagulation reported on his experience of using the INR to prioritise patients with liver transplantation. Hyung Kyung Kim then reported on attempts to standardise PT results with an INR scale as a number of laboratories were no longer reporting PT in seconds, as recommended in the DIC template. She proposed alternative calibration using plasmas from patients with DIC to provide better comparability of INR among different thromboplastins in patients with DIC. The work had the backing of the SSC to continue and a manuscript would be prepared.

Tina Dutt reported on the collaboration with SSC in Plasma Coagulation Inhibitors and UK NEQAS on protein C and antithrombin measurements. As both are increasingly indicated as biomarkers in sepsis trials and treatment, it was the role of the SSC to ensure that measurements are reliable, accurate and consistent between centres. A multicentre evaluation exercise involving plasma from 4 pools of septic patients will be sent to approximately 20 laboratories together with the international standards and congenital deficiency samples. Participating centres will be requested to complete questionnaire detailing conditions such as assay

15 method/normal range establishment. Timeline-Sample distribution in summer 2008, collection by Dec 2008, followed by data evaluation and feedback at the SSC Meeting in 2009 in Boston.

Jecko Thachil reported on the collaboration with SSC in Fibrinolysis on a common scale for reporting Ddimer. The development of a conversion factor to harmonise the different assays has been proposed. Carl Eric Dempfle reported on behalf of the SSC on Fibrinolysis that standardisation of Ddimer was close. As there was no data to inform this SSC on how Ddimer estimations in DIC could be harmonised, the Chair had the backing of the forum to review progress with the Chair of SSC in Fibrinolysis.

Francoise Dignat George from the SSC in Vascular Biology presented on the collaborative efforts with Nigel Key, co chair of this SSC, on measurements of procoagulant microparticles. The first step was to establish a normal range for microparticles and a call would be made for laboratories to participate.

Submitted by C-H Toh

Registry of Exogenous Hemostatic Factors

3 July 2008 Vienna, Austria

Chair: Mary Ann McLane, USA Co-Chairs: Ken Clemetson, Switzerland, Manjunatha Kini, Singapore, Frank Markland Jr, USA, Neville Marsh, Australia, Takashi Morita, Japan

In attendance were Chair McLane, Co-Chair Kini and 6 guests. Co-Chairs Marsh, Markland, Morita and Clemetson were absent with apologies.

1. Meeting was brought to order by the Chairman Dr. Mary Ann McLane. 2. The minutes of the last meeting were read and there were no questions. 3. Dr. McLane reported that the “classification of disintegrins” paper presented at last year’s SSC meeting has now been published with a unique arrangement between JTH and Toxicon: the original paper is now included on the ISTH website, linked through the JTH journal, and is therefore able to be updated as the discovery of new disintegrins proceeds in the future. Due to the nature of the interested readership for this paper, the editor of Toxicon agreed to include a slightly modified abstract in an issue of Toxicon along with a link to the ISTH website and JTH article. This is the first time this has been done by JTH and Toxicon, according to SSC Chair Nuala Booth, and our registry is grateful for the cooperative efforts made to have the widest possible readership for this paper. 4. Members of this registry have reviewed the paper by Drs. Clemetson and Morita on the “Classification and nomenclature of C-type lectin related proteins” and it has been revised based on those comments. It is now ready to be sent to the SSC for final approval and publication, using the same arrangement between JTH and Toxicon as was used for the McLane disintegrin paper described above. Dr. McLane will facilitate its speedy publication once she receives the final copy. 5. Dr. Kini presented new data on anticoagulants from animal venom: hemextin (FVIIa inhibitor) from Hemachatus haemachatus (cobra snake), naniproin (prothombinase complex inhibitor) from Naja nigricollis (cobra snake) and variegin (thrombin inhibitor) from Ambylomma variegatum (tick). 6. Dr. McLane reported that the next classification work for this registry will be for the snake venom metalloproteases, undertaken by Drs. Ana Moura De Silva (Brazil) and Jay Fox (USA). Progress on this endeavor will be reported in 2009 in Boston. 7. Organization of the Fourth International Conference on the Exogenous Factors Affecting Thrombosis and Hemostasis as a satellite meeting to the XXII World Congress to be held in Boston, USA: Dr. McLane, Chairman of the Organizing Committee presented an update to the initial report made in Geneva last year. A contract has been signed with the University of Massachusetts-Boston for the use of their conference center for Friday evening, July 17 through Sunday evening, July 19, 2009. Posters and a reception will happen on Friday, and the next two days will be filled with scientific presentations. This meeting will include acknowledgement of its being the 25th anniversary of the first meeting on Exogenous Factors in San Diego in 1985. The need for widespread advertising of this meeting was emphasized, with a website already in place (http://www.udel.edu/medtech/mclane/EFATH09.html). There is a good possibility of using the Doubletree Bayside (close to UMass-Boston site and to the subway system) as a hotel for participants of both the ISTH and this EFATH09 meeting. Depending on registration numbers and vendor support, we hope to offer travel stipends for students and speakers, as well as travel vouchers while in Boston for all attendees. A tentative listing of speakers, suggested by all registry co-chairs, was made and McLane will begin sending out invitations by August 1. This will include speakers for an ISTH talk, for the SSC educational program (see #10c below) and for the EFATH09 meeting. A proceedings publication will also be published. 8. Other activities: Dr. McLane asked the subcommittee members for suggestions/ concerns in any other areas of exogenous factors. She also encouraged participation in planning and publicizing the EFATH09 meeting. 9. The next SSC meeting of the Registry will be held in Boston, either 11 or 12 July, 2009. 10. Any other matters: (a) Dr. McLane reported on the SSC chairs’ dinner meeting from July 2, 2008: Anna Folanga (Italy) has been elected new SSC Secretary/Chair-elect. Gerhard Johnson will assume the chairmanship after this meeting, succeeding Nuala Booth.

(b) The Cairo SSC meeting will be May 22-25, 2010, with a format similar to this Vienna meeting. (c) The format for the 2009 SSC meeting will be experimental in that subcommittees are encouraged to hold 60-90 minute educational presentations either at the beginning or end of their 4-hour meeting time. These sessions will be listed by topic and speaker as all other ISTH sessions, and this will hopefully encourage wider knowledge and involvement in SSC activities by non-members. Dr. McLane already has informed Bob Hardin, SSC organizer for the 2009 meeting, of our Registry’s interest in doing this. We need to provide topic and speaker before the SSC Executive Committee meets in September. (d) As there were no other matters, the meeting was adjourned.

Submitted by M.A. McLane

Factor VIII and Factor IX

4-5 July 2008 Vienna, Austria

Chair: A. Srivastava (India) Co-Chairs: C. Lee (UK), H. M. van den Berg (The Netherlands), C. Negrier (France), F. Peyvandi (Italy), J.-M. Saint-Rémy (Belgium), C. Hay (UK), J. Oldenburg (Germany), E. Tuddenham (UK)

The Chair opened the meeting at 14:00 and welcomed the audience of about 200 attendants. He explained the structure of the program mentioning that apart from standardization issues in each of the four sections of the agenda, there was one review lecture intended as a CME. He also informed the changes in the presentations.

Completed/Submitted reports and recommendations

There were no completed reports, recommendations or publications to mention this year.

Section I - Rare bleeding disorders Chairs: F. Peyvandi (Italy) and C.A. Lee (UK)

Overview. Flora Peyvandi

Since its inception in 2004, the SSC working party on Rare Bleeding Disorders (RBDs) has attempted to improve the understanding of prevalence, diagnosis and treatments of RBDs by developing the Rare Bleeding Disorders Database (RBDD, www.rbdd.org). At present, the RBDD contains clinical, phenotype, genotype and treatment data of more than 350 patients coming from 21 countries and collected in the last 10 years at the Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre in Milan. The RBDD project has already obtained the collaboration of 66 centres from all over the world that are willing to provide epidemiological information on over 3,000 RBD patients. Recently, data pertaining to two specific projects, designed to answer to particular issues of RBDs, were published online (http://www.rbdd.org/studyonline.htm): a study on menorrhagia and other gynaecological problems in women affected by bleeding disorders and a report on the prevalence of central nervous system bleeding and their therapeutic approach in patients affected by RBDs. To further improve the quality of data being obtained from these centres, there is a need for a good communication tool that will allow inserting, managing, editing and viewing information on RBD patients. The development of such a toll was made possible by a recently funded European project (EN-RBD network: http://ec.europa.eu/phea/documents/2006_Health_Information.pdf), in which 10 centres would collaborate (www.rbdd.eu). In November 2007, the first workshop of the EN-RBD working group was held in Milan. The testing phase, where partners started to insert specific data about their severely affected patients is on-going. By the end of June 2008, ~10% of patients records were inserted. Queries and reports allowed us to derive preliminary results about clinical manifestations, treatment and identified mutations (www.rbdd.eu). In conclusion, the on-line database resulted to be a powerful tool to improve the quality of data collection.

Defining clinical severity of rare bleeding disorders. Flora Peyvandi

The rare bleeding disorders (RBDs) are associated with a wide spectrum of clinical bleeding manifestations. In order to understand the correlation of bleeding with the level of factor deficiency, it is necessary to have tools to quantify clinical bleeding in these patients. Recently, two different tools were proposed to quantitatively bleeding history. The bleeding score proposed by Rodeghiero and Tosetto et al, principally applied to type 1 von Willebrand Disease (VWD) patients, summarises both the number of episodes and their severity. However, this score was validated only in type 1 VWD and, in a small study, on type 3 VWD obligatory carriers. The bleeding score (BS) proposed by Srámek et al, and modified by Podda et al, accounts for the number of bleeding symptoms and their nature, severity, duration, localization, frequency. This score was only tested in 128 patients with suspected abnormalities of haemostasis, where a BS >12 was associated to severe bleeders. Unfortunately, up to now, none of these scoring systems was specifically applied to RBDs. We tested them on a group of 35 women affected by RBDs. The results showed that both the scoring systems are good to

19 distinguish even mild RBDs patients from controls; however, there was no significant differences in the bleeding scores of patients with different levels of phenotype severity. The mean BS increased from patients with a mild to those with a severe deficiency in the analysis of the only women affected by FVII deficiency (n=14), whereas an opposite trend was observed in a small group of women affected by FXI deficiency. Tools for quantifying clinical bleeding need to tested and validated in people with RBDs. The assay methods for measuring these factor levels need to be standardized and their quality assessed.

Defining Severity for Rare Factor Deficiencies: Insights from the NARBDR. D. DiMichele, S. Acharya

The FVIII/IX Subcommittee posed two questions: How can disease severity be defined for rare bleeding disorders (RBDs)? Could the North American (NA) Rare Bleeding Disorders Registry (NARBDR) database (Acharya, JTH,2004) offer potential answers to this question?

Among 284 subjects in the NARBDR, 78% had FII, FV, FVII, or FX deficiency for which we could not establish homozygosity / heterozygosity on the basis of molecular studies because only 5.4% subjects underwent gene- based diagnosis. Factor activity levels, available for 96% subjects, proved more useful. Based on a similar strategy by Roberts (T&H, 2003), ³ 0.20 u/ml (“hemostatic”) factor activity was originally used to define heterozygosity for between 40 and 60% of each RBD. However, since other RBD registries had published alternative severity schema, the investigators performed a reanalysis of the original dataset to correlate FII, FV, FVII, FX activity levels with: age at first bleed, bleeding trigger and site of bleeding. Bleeding site / trigger data were examined according to two-disease severity schema: mild disease was defined by either the original NARDBR designation (³ 0.20u/m) or the more traditional hemophilia model (³ 0.05u/ml). The NARBDR provided limited but interesting data on the association between bleeding parameters and disorder severity as ascertained by factor activity. The authors concluded that: 1) Rare deficiencies may not be amenable to a single severity categorization as attested to the differences between the Vitamin K- dependent factors and FV; and 2) Similar analyses of larger more informative national and multi-national RBD prospective databases will be useful in defining clinical severity for these disorders.

Factor assays in patients with rare bleeding disorders - Data from UK NEQAS. S. Kitchen

Data from clotting factor assays of FII, V, VII, X and XI performed in 230 to 330 laboratories participating in the UK National External Quality Assessment scheme (UK NEQAS) are discussed. Lyophilised samples from the same donor were analysed using whatever method was in local use. The CVs of results for samples from normal subjects were between 9 and 17%. For samples with levels of 20 - 27 IU/dl CVs were higher at between 23 and 39%. These figures are similar to those obtained by the same laboratories when performing assays of FVIII and IX in both groups (normals and mild deficiency). The variables which contribute to this imprecision include the use of normal plasma unit in the case of FV and FXI where the IU has not yet been adopted by a number of manufacturers of reference plasmas used for assay calibration. Some variability occurs as a consequence of use of different reagents, for example in the case of different thromboplastins in FVII assays. A major contributor to assay variability is the use of poor assay design which often does not follow recommendations available in the literature. Regular external proficiency testing of assays for rare bleeding disorders should help to draw attention to these problems and educate centres in the need for more standardised assay design.

Replacement of the 3rd International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma, Human. E. Gray

The current WHO 3rd International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma, Human, NIBSC code 99/826 was established in 1999. The stock of this standard is expected to be exhausted by December 2010. This is an important primary standard used not only by clinical laboratories and manufacturers of plasma derived blood products, but also for calibration of secondary standards such as the SSC/ISTH Secondary Coagulation Standard. A replacement standard is essential for the continuity of units of the four coagulation factors. The value assignment of the replacement standard will involve assays of all four factors against the current 3rd International Standard and locally collected normal pooled plasma. As the SSC/ISTH Secondary Coagulation Standard Lot#3 will also require replacement at approximately the same time, it is envisage that this exercise will also include the calibration of the SSC/ISTH Secondary Coagulation Standard Lot#4. The collaborative study will be initiated in September 2009 with a final study report

20 submission date to SSC in July 2010. Upon approval by the SSC, the proposed candidate will be recommended to the Expert Committee on Biological Standards for establishment as the 4th International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma, Human in October 2010.

Replacement of the WHO International Standard for Factor VIIa Concentrate. A. Hubbard, L. Weller, A. Heath

The current WHO 1st International Standard (IS) Factor VIIa Concentrate (89/688) is used for the potency estimation of activated factor VII (FVIIa) concentrates intended for therapeutic use and also for FVIIa estimation in plasma and prothrombin complex concentrates. Stocks of the WHO 1st IS are extremely low and a replacement preparation is required. Calibration of the replacement WHO IS has been achieved through an international multi-centre study involving 23 laboratories. Two candidate preparations (sample B: recombinant and sample C: plasma-derived) were assayed directly relative to the WHO 1st IS (sample A) using the conventional one-stage clotting method (175 assays), the chromogenic method (30 assays) and the clotting assay specific for activated factor VII using soluble recombinant tissue factor (21 assays). Estimates by conventional clotting assays using a common “provided” thromboplastin reagent and local thromboplastin reagents were extremely similar and gave combined mean values of 655.63 IU/ampoule (n=43) for sample B and 679.77 IU/ampoule (n=43) for sample C. For sample B, estimates by the chromogenic method and activated factor VII clotting method differed by 1% and 2% respectively from the conventional clotting method and this emphasises the similarity of sample B with the WHO 1st IS which were both prepared from the same product. In contrast for sample C, the estimates by the chromogenic method and activated factor VII clotting method differed by 7% and 22% respectively from the conventional clotting method. Estimates of stability based on accelerated degradation studies, after 6 months storage, together with long-term experience with other FVIIa preparations, indicate that the candidates should be very stable when stored at -20 °C. In the interests of continuity and, in consideration that sample B represents the only licensed Factor VIIa therapeutic concentrate available at the present, it is proposed that sample B (NIBSC code 07/228) is accepted as the WHO 2nd IS Factor VIIa Concentrate with an assigned potency of 656 IU/ampoule.

Licensure of products for therapy of rare bleeding disorders - EMEA perspective. R. Seitz,, G. Silvester

In the European Communities (EC), products are available for rare coagulation disorders, e.g. Fibrinogen, FVII, FXIII, but still some are missing. The European Orphan Medicines Legislation provides incentives, such as free protocol assistance (scientific advice), fee reductions, and market exclusivity for 10 years, as well as EC programs to support research, development and availability. In the area of blood products, some new and follow-on products have been designated. The EMEA has also installed an office for support of small and medium-sized enterprises (SME). There is the possibility of parallel EMEA and FDA scientific advice meetings. There are regulatory mechanisms to license with limited clinical data: Conditional Authorisation and license under Exceptional Circumstances, where the applicant can show that he is unable to provide comprehensive data on efficacy and safety. Another important mechanism is Compassionate Use. An example is Protein C, where in response to requests from physicians, after years of compassionate use, the product was authorised in May 2001 under ’Exceptional Circumstances‘, and after the company supplied additional information ’Exceptional Circumstances’ ended in July 2006. The CHMP Blood Products Working Party (BPWP; Chair R. Seitz) elaborates guidance on clinical assessment of products. Since not enough patients are available to perform statistically meaningful pre-licensing studies, it is understood that pre-licensing clinical studies alone will not provide full assurance of safety, and post-marketing studies and registries play an important role. How can we obtain more products in the EC? We need the demand expressed by physicians and/or patients to have access to products which are so far lacking, to motivate companies to develop a products taking advantage of the available regulatory assistance (e.g. orphan medicine designation, protocol assistance) throughout the licensing process.

Licensure of products for therapy of rare bleeding disorders – US FDA perspective. M. Weinstein

FDA promotes the development of products that demonstrate promise for the treatment of rare diseases. Generally, the disease or condition for which the candidate orphan drug is intended affects fewer than 200,000 people in the United States. Incentives available to encourage orphan drug development are 7 years of market exclusivity following approval of an orphan drug; 50% tax credit on certain clinical testing expenses; clinical study grants; and waiver of the Prescription Drug User Fee Act application fee. Adequate and well controlled

21 clinical trials are necessary to demonstrate the safety and efficacy of orphan drug products. FDA will work with sponsors to develop clinical trial protocols that are appropriate for the size of the patient population. A recent example is the approval of Protein C Concentrate (Human), a human plasma derived product. Approval was based on treatment of acute thrombotic episodes including purpura fulminans (PF) in an open label non- randomized study in 18 subjects with severe congenital Protein C deficiency compared to the efficacy ratings for 21 episodes in an historical control group. FDA also encourages international cooperation in orphan product development through such programs as the EMEA – FDA parallel scientific advice meetings. This continuing program, initiated in 2004, provides a mechanism for EMEA and FDA to exchange their views on scientific issues during the developmental phase of new medicinal products. This program can optimize product development, and avoid unnecessary testing replication or diverse testing methodologies. FDA is also interested in establishing and using patient registries and databases to increase understanding of the rare plasma protein disorders. Registries and databases have the potential to help in product development; to make available information about the natural history of the disease; to provide guidance in dosing and frequency of dosing; and to aid in surveillance, and adverse event reporting.

Section II - Factor VIII.IX: Clinical issues - I (Assays / EQA / Phenotype) Chairs: C. Negrier (France) and H.M. van den Berg (The Netherlands)

Phenotypic heterogeneity of severe hemophilia – Defining the issues. A. Srivastava, India

It has been long recognized that 10-15% of patients with ‘phenotypically characterized’ severe hemophilia (<1% clotting factor activity) have relatively mild disease clinically. Not all these patients have frequent spontaneous bleeding and even among those who bleed, the extent of joint damage tends to vary considerably. The basis for this difference has not been completely understood. Clinical observation and laboratory data from animal models suggests that this heterogeneity exists at several levels. Differences in overall hemostatis efficiency could account for the some of the differences in bleeding frequency. Preliminary data from tests of global hemostasis carried out in this population suggests that these tools can be informative but further evaluation is needed particularly among patients with <1% FVIII:C but clinically heterogeneous disease. The second levels of heterogeneity proably occurs at the level of the inflammatory response to the iron in the synovium. With the central role of hemosiderin deposition in initiating inflammation in the joint following hemarthroses has well documented in animal models, it is tempting to predict that functional polymorphisms in the immunomodulatory cytokines could affect this response. In fact we have some early data from patients with minimally treated severe hemophilia to suggest that this could be so. Finally, the extent of synovial hypertrophy could be affected by polymorphisms in the cytokines affecting angiogenesis. This needs further evaluation. One of the major issues in the management of hemophilia today is to decide on ways in which therapy, particularly the initiation and intensity of prophylaxis, can be individualized. A detailed understanding of all factors that may contribute to joint damage in severe hemophilia could help us in tailoring therapy for these individuals.

Thromboelastography – An International Study on reproducibility and consistency. M. Chitlur

Thromboelastography is a global assay of haemostatic function, from the beginning of clot formation to fibrinolysis. Two systems utilizing this technology include Thrombelastograph/ TEG® (Haemonetics, Braintree, Massachusetts, USA) and ROTEM (Pentapharm GmbH, Munich, Germany). They are simple to use and proven to be an effective methods of intraoperative monitoring to optimize blood product selection and utilization and improve outcomes during complex surgical procedures such as cardiothoracic surgery and liver transplantation. Case reports or small case series have addressed the usefulness of thromboelastography in the management of bleeding or monitoring of treatment with bypassing agents. In order to establish the usefulness of this test a standardized methodology needs to be developed and reproducibility and consistency have to be demonstrated. In an attempt to initiate this process, the TEG-ROTEM Working group was established in 2006 and investigators from several countries joined hands to perform studies. Here we present the results of the first international study, wherein 9 laboratories from 6 countries (Canada, Denmark, Israel, Norway, UK and USA) participated. Standard pooled normal plasma as well as FVIII deficient plasma was activated using either kaolin (TEG) and INTEM (ROTEM). Clotting parameters i.e. clotting time (R/CT), time to clot formation (K/CFTR), Maximum clot firmness (MA/MCF), clot strength (G) and rate of clot polymerization (Angle) were measured. The CV’s varied from 6 to 60% and varied significantly for the different parameters, with the lowest CV’s being seen with the R/CT and MA /MCF. The inter laboratory variance was also significant

22 with CV’s greater than 10%. Even though these results are not satisfactory, this has been the first effort to standardize this methodology and significant work remains to be done to improve the reliability and reproducibility. These studies were performed on PRP and the results may be more reliable when preformed on whole blood samples. Future studies will use whole blood; determine the effect of anticoagulants and the effect of different activators on the clotting parameters, helping us to optimize the methodology and improve the reliability characteristics.

ROTEM – Usefulness in predicting response to bypassing agents. E. Tuddenham

Thromboelastography has been proposed as a means to measure and predict response to bypassing agents ( rVIIa and aPCC). In our laboratory we have investigated in-vitro addition of bypassing agents to whole blood from patients with FVIII or FIX antibodies. Blood is taken into citrate with corn trypsin inhibitor and coagulation is initiated with CaCl2 and 1/50,000 ( ~1pm) dilution of rTissue Factor. Clot stiffness was measured with ROTEMR . In three cases subjected to surgery the predicted effect of invitro addition of bypassing agents was confirmed by adequate haemostasis during procedure. Six further cases were also tested but have not yet undergone surgical challenge. Three patients had normal ROTEM parameters prior to addition of bypassing agent. Further studies with intraoperative (ex vivo) analysis are needed to validate the assay for its clinical usefulness, but results so far are encouraging.

Thrombin generation test - Usefulness in monitoring therapy. C. Negrier

Since haemostasis is considered to be a combination of multiple proteins which can assemble in complexes at the cellular surface, global evaluation of this process may be more relevant in some situations. Thrombin generation assay (TGA) is one of the candidate which may theoretically be used to further precise the phenotype of a particular patient and to measure in vitro and ex vivo the effects of bypassing agents used for the treatment of inhibitor-developing haemophiliacs. We have established at our institution the normal range for various parameters of the TGA and have demonstrated that these parameters were significantly impaired in patients with various severities of haemophilia A and B. The TGA parameters were completely normalised upon substitution with factor VIII or factor IX, but needed more subtle dose adaptation when bypassing agents were used. Because of the inter-individual variations in terms of biological response to bypassing agents, we have designed a 3 step-protocol where both the therapeutic agent and the dose are chosen according to TGA results for challenging situations such as planned surgeries. High risk surgeries have been conducted in consecutive patients with results that matched the predictions of the in vitro and ex vivo assays performed pre- operatively, suggesting that TGA may be useful to predict the effective dose of bypassing therapies and to follow up the haemostasis profile during and after the surgical procedure. Our results strongly suggest that this assay could represent a surrogate marker for monitoring bypassing therapies in surgical situations, and this approach needs further validation. A working party is proposed with the aim to correlate the results from the assay with clinical parameters.

External Quality assessment of ROTEM. S. Kitchen, D.P. Kitchen

Data were presented from 3 external quality assessment surveys through the UK National External Quality Assessment scheme. These included results from 14 users of TEG and 10 users of ROTEM who analysed lyophilized plasma samples from normal subjects and patients with severe FVIII and FXI deficiencies. Each sample was analyzed once as per routine practice. The CVs for Extem and Intem tests on the ROTEM were 25%-121% and 10% – 37% respectively. Clot firmness was better for the sample with 5g/l fibrinogen than those with lower levels. The CV of results on the TEG were in the range 15 -33%. The data indicated that plasma could be successfully used for EQA purposes and it was possible to identify centres with outlying results.

Assay discrepancies between one and two stage assays – a)Discrepant assays in mild haemophilia. S. Kitchen, M. Makris

Three main methods are available for FVIII:C activity estimation. The one stage clotting assay is used by the vast majority of laboratories internationally, whilst a small number use the chromogenic and two stage clotting assays. All of these assays give equivalent results in severe haemophilia and following most FVIII replacement.

In mild haemophilia however, where the structure of the molecule is abnormal, significant differences between the assays are seen in some cases. In Sheffield, UK we use the two stage clotting assay as our standard assay. We have recently started re-examining all our mild haemophiliacs and found significant discrepancy in around 30% of our patients. The two stage clotting assay results were largely similar to chromogenic results. We defined discrepancy when the ratio of the one stage: two stage results were outside the 0.5-2.0 ratio. From 56 mild haemophiliacs we identified 10 with discrepancy in which two stage levels were significantly lower than one stage ones. Seven (12% of the study group) of these patients with definite bleeding histories and genetically confirmed haemophilia had FVIII:C that were normal by the one stage assay and the diagnosis of haemophilia would have been missed. 6 patients (11%) in our study group had FVIII:C that were reduced by one stage but normal by two stage assay. With referrals we have now identified 16 individuals from 10 families with this pattern all of whom had the Tyr346Cys mutation and largely negative bleeding histories. In all our patients chromogenic assay results are broadly in agreement with the 2 stage clotting method. In the Sheffield experience, the two stage clotting assay reflects the severity of haemophilia better than the one stage assay which can fail to diagnose some and over diagnose other patients as mild haemophiliacs. b)FVIII assay discrepancy - Hemophilia A variants escaping the two-stage assay. H. Brondke, J. Oldenburg

Factor VIII activities are mainly determined by either the one-stage clotting assay or by a two-stage chromogenic assay. Although these tests give similar Factor VIII activities in the majority of hemophilia A patients, a small subset of the currently known mutations are characterised by discrepant results in these assays. The larger fraction of these mutations lead to higher FVIII concentrations in the one-stage assay, while the two-stage assay displays an activity, which is congruent with the clinical picture of the patients. However 6 mutations (Glu321Lys, Tyr346Cys, Ile369Thr, Glu720Lys, Arg1689His and Phe2127Ser) show a reverse effect, in which the two-stage assay is higher than the one-stage. In these, mainly mild hemophilia A cases, the effect of the underlying mutation is only apparent in the one-stage clotting assay, as the two-stage assay yields normal activities. A common feature of the above mentioned mutations, except for Phe2127Ser, is that they cluster in the vicinity of thrombin cleavage sites.

Section III - Factor VIII/IX: Clinical issues – II: Inhibitors Chairs: C. Hay (UK) and J.M. Saint-Remy (Belgium)

Biology of the development of inhibitors: Current understanding focusing on B cells. JM Saint Remy

The bone marrow produces millions of new B cells every day with high risk of producing B cells capable of reacting with FVIII. FVIII exposure is by definition a recurrent event, driving B cells into process of affinity maturation. The B cell pool at one given moment in time is mode of very heterogeneous population of cells with different sensitivity to signaling events. The purpose of this short review is to discuss the ways by which B cells interact with their environment and to present a clearer understanding of how this could be manipulated for the benefit of patients with inhibitors.

B cells interact with their environment via 3 types of receptors: the antigen-specific receptor (BCR), the CD40 molecule and Toll-like receptors (TLR). The CD40 molecule allows B cells to interact with T cells via CD40 ligand and will not be considered here. The BCR is coupled to a complex network of intra-membrane and intracellular factors, the aim of which is to transmit surface activation into metabolic events. Depending of the stage of maturation of the B cell, the BCR-mediated events will depend on the format of the ligand (FVIII), the BCR avidity and the localization of the B cells. TLRs bind pathogen-derived factors and the consequences of TLR binding vary dramatically from cell to cell. Thus, for a same stimulus, B cells and dendritic cells will react in opposite directions. This offers possibilities to manipulate B cells without touching upon the normal capacity of the immune system to react to pathogens. The molecular events that are decisional for B cells to enter either in an differentiation process to produce antibodies or to recycle within germinal centers are well established. At transcriptional level, BLIMP-1 is a master regulator, which, upon activation, drives B cells into plasma cells. Numerous factors, some of which can be manipulated, either activate or suppress BLIMP-1 transcription, providing another stage at which the production of inhibitors could be counteracted. The recent generation of a BCR-transgenic mouse strain makes it possible to evaluate at molecular level every step leading to activation and/or suppression, thereby providing a new tool to explore therapeutic approaches for eradication of inhibitors.

Analysis of inhibitors in clinical trials: Where are we now? N. Jain

During the 2003 FVIII inhibitor workshop sponsored by FDA, problems related to evaluation and assessment of inhibitors in FVIII deficient patients were identified. These were: definition of an inhibitor, cut off value of a positive inhibitor, definition of high and low inhibitor and clinical implications of a transient inhibitor. In order to obtain uniform data on inhibitor developments in a clinical trial, FDA recommends a standard way of analyzing inhibitors in a clinical trial(s). Based on recent publications on difference in rate of inhibitors between pd and recombinant products, FDA investigated its AERS database. No conclusion could be drawn on rates of inhibitor development between pd and rFVIII products because of the limitation of the above database. FDA has attempted to collect a uniform data on inhibitor development during the development phases of a new and /or product undergoing major manufacturing changes.

Rodin study update. H. Marijke van den Berg

Observational cohort studies are an important design to investigate the etiology of inhibitor development. A large number of genetic factors like large gene mutations of the factor VIII gene have been recognized as important factors. Apart form the genetic factors, treatment related factors have an impact in inhibitor development. There is still a large debate on a different impact for plasma or recombinant products on inhibitor development. Recently in the Canal study the large influence of a peak treatment moment on inhibitor development have been clearly demonstrated. Patients that were treated at their first treatment episode with a peak moment had an increased relative risk of 6.8 that they were going to develop an inhibitor. In contrast patients that started with early prophylaxis had a decreased risk to develop an inhibitor. The hypothesis is that danger signals like intensive treatment and infection or surgery are mechanism that can induce inhibitor development in a particular patient. This also is an explanation why for instance in identical twins there can be discordant inhibitor development. In the Rodin study data are collected from all children with severe hemophilia A born between 1-1-2000 and 1-1-2008. Patients will be followed until they have received 75th exposure days. In structured base line and follow-up forms every reason for treatment, the dose, mode of infusion etc will be captured along with data on vaccinations, infection and surgery. At the moment 28 centres have IRB approval and already >270 patients with severe haemophilia A have been included. The estimated number of patients to be included is a total of at least 800. More information on the study can be obtained on the www.rodinstudy.eu.

Update: International ITI Study. D. DiMichele

D. DiMichele presented an update on the International ITI Study on behalf of co-PI, Charles Hay, and the I-ITI study group. The study is a prospective randomized multi-center trial of the safety and efficacy of high (200 u/kg/d) vs. low (50 u/kg thrice weekly) dose ITI in a good risk cohort of high titer hemophilia A inhibitor patients. The data for the cohort as a whole was presented; investigators remain blinded to interim arm-specific results. To date, 106 of the targeted 150 subjects have been enrolled from 96 centers in 25 countries. As of 4/08, 91 have been randomised at a median age of 23.4 months) and a median pre-ITI titer of 5.0 BU (0.6 - 9.4), achieved after a median 5.0 (0 – 23) months from inhibitor diagnosis. This cohort has been on ITI for a median 13.0 (0.8 – 33.0) months. To date, 44/91 achieved negative titers; 38 achieved normal FVIII recoveries; and 35/91 became tolerant after a median of 14.9 (3.5– 30.4) mon ths from ITI start. Forty-three subjects have reached a study endpoint; a total of 11 subjects have failed ITI, based on one of two failure criteria. Two interim data safety analyses are mandated by the protocol when 50 and 100 subjects completed the study. In May 2008, the DSMB conducted its first in camera interim comparative analysis of study treatment arms with respect to safety and efficacy, and concluded that it was safe to continue the trial. Independent investigators are conducting 7 satellite studies in conjunction with the trial. The PIs continue to welcome any additional participating centres until target enrolment is achieved.

Harmonisation of National Databases for investigation of Inhibitor development in Haemophilia A. C.R.M. Hay

CHMP and EMEA requested ISTH to facilitate harmonization of data collection by national databases to enable them to share anonymous data. This would permit analysis of large numbers of patients to investigate risk- factors for inhibitor development in previously treated patients (PTPs). Possible risk factors for investigation include treatment factors such as product switching, product type, treatment pattern use of immunomodulatory therapy and intense treatment episodes and surgery, and host factors such as factor VIII genotype, age of the

25 patient, ethnicity and first degree family history. For such data to be aggregated in the form of a meta-analysis, the contributing databases would have to collect similar data using similar methods from a similar sample of their patient population. This process aims to achieve adequate harmonization of data collection to make such collaboration a practical proposition. To this end, data-fields have been exchanged and conference calls conducted and a detailed questionnaire circulated between those running the UK National Haemophilia Databases and the National Databases in the USA, Germany, Italy and Canada. Informal discussions have also taken place with several other databases and it is hoped that they may also become involved in this process. The emerging European Adverse Event Surveillance System (EUHASS) has also been involved in this discussion but may not be able to participate because it has a different model and considerable overlap with several other European networks.

All the other participating databases aspired to be complete National Networks though country coverage was incomplete for the US and Italian networks. The German database is only currently being established and is too new to evaluate from this perspective. Participation is voluntary in each case, with some central governmental compulsion in the UK and Germany. Patient consent was required by all countries but Canada, though all databases but the UK database had varying degrees of patient anonymity, usually falling short of the requirements of the European Data Protection legislation. Only three had a national network although all but the Canadian Databases used similar or identical software and hardware organization. Staffing varied in different countries. The UK and US databases were well staffed but all the others were run by one or two people, raising doubts about data quality and capacity to collaborate. With the exception of the Canadian database, there was a very high degree of harmonization of data collected, though all databases were at an early stage in collating genotypic data. All databases committed informally to the principle of sharing aggregated anonymous data according to a pre-agreed protocol. Merging of national databases is not anticipated and has not been discussed. Further conference calls and meetings will be arranged to push the process forward.

Role of Immune Response Genes in Inhibitor Development. J.Oldenburg, J. Astermark, E. Berntorp

The formation of alloantibodies against factor VIII (FVIII) or factor IX (FIX) is the most severe complication of replacement therapy in patients with haemophilia. In the last decade, genetic factors have been shown to constitute a decisive risk determinant for the development of inhibitors. In severe haemophilia A and B, mutations that result in an absent or truncated FVIII/FIX protein are associated with a 20–80% risk of inhibitor formation. Missense mutations represent the main mutation type with a low inhibitor prevalence of 5%. Recently, a significant association between inhibitor formation and polymorphisms in genes coding for cytokines (IL-10) and other immunoregulatory factors (TNF-a) has been shown. These genetic factors constitute the individual genetic risk profile of a haemophilic patient. This risk is imprinted and fixed; however, environmental factors such as treatment schedule may increase or decrease the inhibitor risk in an individual patient. Improved understanding of these complex interactions may lead to the development of preventive measures to minimize inhibitor formation. Why some haemophilic patients develop inhibitors while others do not and whether it may be possible to predict their development, are two major issues that still need to be resolved. However, a genetic predisposition to inhibitor development to FVIII/FIX proteins is clearly present. In part, this vulnerability depends on the underlying FVIII/FIX gene defect (inversions, nonsense mutations, etc.). For most gene alterations in haemophilia A, the inhibitor risk ranges from 20% to 40%, corresponding to an OR of about 2, while for some FVIII gene defects, the OR can be 10–30 (nonsense mutations vs. missense mutations and large multidomain deletions vs. missense mutations). However, immune response genes may play a decisive role in inhibitor formation, as has been reported for IL-10 and TNF-a, and may prove to be even more important than the FVIII/FIX gene defects. The ongoing challenges are to fully discover and characterize these variables and their interactions with environmental factors, and then to apply that information to prevent inhibitor formation, thus overcoming this most serious complication of haemophilia treatment.

Section IV - Factor VIII/IX: Standardization issues Chairs: E. Tuddenham (UK) and J. Oldenburg (Germany)

Factor VIII at 4.5 Angstroms - Insights from the new crystal structure. E. Tuddenham

Two papers had been published in the last 6 months reporting medium resolution X-ray crystallographic structures of B domain less rFVIII. (Structure. 2008 Apr;16(4):597-606; Blood. 2008 Feb 1;111(3):1240-7) The

26 structures are very similar and differ from earlier low resolution electron diffraction structure mainly in the position of the C2 domain, which is side by side of the C1 domain. Two Cu++ atoms are present in the A1 and A3 domains and two Ca++ atom are also visualized. Three carbohydrate side chains were also well visualized even though partly buried. PDB coordinates for FVIII are available online to download and use for interpreting mutations, antibody epitopes and novel FVIII designs, as well as many other applications.

SSC - Factor IX concentrates “Field” studies: A new proposal. S. Raut, M. Lee

Previous SSC “Field” studies on FIX concentrates have shown high inter-lab variability [CVs 9% - 23%; TG tests ~110%]. Recent SSC “Field” studies on FVIII concentrates have shown that high inter-laboratory variability can be significantly reduced by following SSC assay recommendations. The last FIX study was carried out 1997 and since then a number of (new generation) recombinant FIX concentrates are under development (Phase I/II Clinical Trials) and it is important to ensure correct potency measurement of these new and older FIX products. Following discussion at meetings of the SWP on field studies (ISTH/SSC FVIII/FIX Sub-Committee), it was decided to investigate causes of variability observed. Two studies are proposed: Phase 1 - Field Study using routine methodologies (to evaluate current status in assaying FIX concentrates); Phase 2 - Controlled Study following strict protocol instructions, to assess the effects of different, methods, pre- diluents, operators and assays on separate days. In addition, components of variation analysis will be carried out by Martin Lee. Four materials are proposed to be included in the studies (2 plasma derived & 2 recombinant). In addition to potency assessments, participants are requested to carry out thrombogenicity tests also (e.g. NAPTT, thrombin/fibrinogen clotting time (TFCT), FIXa etc.) Samples and Protocol for the Phase I study is scheduled to be dispatched in Feb 2009 with participants returning data by 1st May 2009. It is proposed to present preliminary data to the SSC FVII/FIX Sub-committee in July 2009.

Collaborative Study to Establish the 4th International Standard, the EP BRP and the FDA Standard for Factor IX Concentrate. E. Gray, W. Pickering, J. Hockley, P. Rigsby, E. Terao, KH Buchheit, M. Weinstein, T Lee and R. Drews

Thirty laboratories from 14 countries took part in a collaborative study to assign potency values to a World Health Organisation (WHO) replacement international standard (IS) for Blood coagulation Factor IX, concentrate, Human, a replacement European Pharmacopoeial (EP) Biological Reference Preparation (BRP) and a replacement FDA Standard. Three candidates, one of recombinant origin and two of human plasma derived origin, were assayed against the 3rd International Standard for Blood Coagulation Factor IX, Concentrate, Human (96/854). The 3rd International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma, Human (99/826) was also included to evaluate the relationship between the factor IX plasma and concentrate units. Thirty-two sets of clotting assay results and two sets of chromogenic assay data were analysed. There was significant difference in potency estimates by these two methods for sample B, the recombinant candidate and sample P, the plasma IS. Similar potency values were obtained for samples C and D, the plasma derived products by clotting and chromogenic assays. For the clotting assays, intra-laboratory variability (GCV) was found to range from 0.5 – 21.7%, with the GCV for the majority of laboratories being less than 10%. Good inter-laboratory agreement, with the majority of the GCV being less than 10% (GCV range = 4.7 – 10.6 %) was also obtained. The estimated potency of the 3rd IS for plasma factor IX relative to the 3rd IS for factor IX, Concentrate was found to be 6% lower than with the assigned value. This discrepancy appeared to be a carry-over from the calibration of the 3rd IS for factor IX, Concentrate. Considering the preliminary stability data, the intra- and inter-laboratory variability, and the differences between the clotting and chromogenic assay results, it is proposed to recommend that sample C, 07/182, to be established as the 4th International Standard for Blood Coagulation Factor IX, Concentrate, Human (96/854), the EP BRP for Human coagulation factor IX concentrate, batch 2 and the FDA reference standard for Human coagulation factor IX concentrate, with a clotting potency value of 7.9 IU/ampoule.

Factor VIII inhibitor study: Status update: K. Mertens

The development of a FVIII inhibitor standard was initiated at the 2003 Subcommittee meeting. The need for this reference was also endorsed by WHO and, more recently, by EMEA. Dr. Raut (NIBSC) has been running the study, and a progress report of the study has been presented at the SSC meeting in Oslo in 2006. The study had been performed by 22 expert laboratories, and addressed 5 candidate preparations for the International Standard, including a rabbit polyclonal inhibitor, 2 human monoclonal antibodies, and 2 human

27 inhibitor patient plasmas. The study highlighted that although one candidate preparation (inhibitor patient plasma 05/206) had the lowest overall CV (17.7%) with a mean Bethesda titre of 8.2 BU/vial, both inter- and intra-laboratory variability were relatively high (CVs up to 36%). Moreover, when the results were recalculated relative to the 5 candidate preparations, only a slight improvement in inter-laboratory CVs was observed, suggesting that the new standard would be of limited benefit. The progress report has raised numerous concerns, including the viral status of candidate 05/206 (HCV and HIV positive), the high CVs, and uncertainty with regard to statistical validity of assays for residual activity. In 2007, a Working Party was established (Chair: K. Mertens) with the aim to assess the feasibility a ‘post-hoc’ statistical validity check in order to identify the most reliable inhibitor assays. This, however, may be possible for only part of the data. Dr. Mertens discussed several options to proceed from now, ranging from using data from a limited number of participants only, to performing a new, smaller study under more controlled conditions. The audience was invited to send suggestions to Dr. Mertens ([email protected]) for further discussion in the FVIII inhibitor Standardisation Working Party.

Calibration of the replacement WHO International Standard for FVIII/VWF Plasma. A. Hubbard

The current WHO 5th IS Factor VIII/von Willebrand Factor, plasma was established in 2003 with assigned values for FVIII:C, FVIII:Ag, VWF:RCo, VWF:Ag and VWF:CB. With an average despatch of approximately 750 ampoules per year it is expected that stocks will be exhausted in 2009. A candidate replacement preparation consisting of approximately 20,000 ampoules was filled in March 2008. Calibration of the proposed WHO 6th IS will involve assays for all 5 analytes relative to the WHO 5th IS and locally collected normal plasma pools. The former comparison is essential for continuity between standards and the latter comparison provides a check on the value of the IU relative to the “fresh plasma unit”. The collaborative study is scheduled for September – November 2008 with analysis completed early in 2009. The study will be submitted to SSC for endorsement in July 2009 prior to submission for formal establishment by the Expert Committee on Biological Standardisation of WHO in October 2009.

Proposed 8th International Standard for FVIII Concentrate: Update. S. Raut

The current WHO 7th IS Factor VIII concentrate (99/678) was established in 2004 with assigned values for FVIII:C. This standard is used for the potency estimation of FVIII in therapeutic concentrate products, both recombinant as well as plasma derived FVIII, which are used for the treatment of haemophilia. Approximately 600 - 800 ampoules are despatched each year and it is envisaged that stocks of the current WHO 7th IS (99/678) will be exhausted by late 2009. Although the current 7th IS is plasma derived, materials for its replacement have been sourced from product manufacturers of both plasma derived and recombinant FVIII concentrates. Following assessment of numerous trial fills, four candidates (2 plasma derived & 2 recombinant) have been selected and 20,000 ampoules of each have been filled (March - April 2008). Calibration of the proposed WHO 8th IS will involve assays for all 4 candidates (using one-stage APTT & chromogenic methods) -relative to the current WHO 7th IS and BRP batch 3/Mega 2US standards. These comparisons are essential for both continuity and harmonization between the 3 different standards. Furthermore, as the BRP batch 3 also requires replacement, this will be a joint project between NIBSC & EDQM to replace both standards. Participants (35 laboratories) have been recruited and the joint collaborative study has been launched (June 2008) with analysis scheduled to be completed in late 2008. The study for the proposed 8th IS will be submitted to SSC for endorsement in July 2009 prior to submission for formal establishment by the Expert Committee on Biological Standardization of WHO in October 2009.

Collaborative Study on a Candidate International Genetic Reference Panel for Haemophilia A, Intron-22 Inversion, Human gDNA. E. Gray, W. Pickering, M. Hawkins, J. Boyle, R. Hawkins, P. Metcalfe

Fourteen laboratories participated in an international collaborative study to assess the suitability of a panel of four genomic-DNA (gDNA) samples as the 1st International Genetic Reference Panel for Haemophilia A, Intron 22 Inversion, Human gDNA, NIBSC Code 08/160. The panel consists of gDNA from a normal male (06/186), a normal female (06/200), a female carrier (06/204) and an affected male (07/116). The participants evaluated the panel against their in-house controls which were characterised patient samples. In total, 166 genotype tests were carried out on the panel, with an error rate of 1.8 %. The findings of this study indicated that this panel is suitable to be used as a reference material for genotyping of Haemophilia A intron 22 inversion mutation. The participants all agreed with the recommendation that this panel (08/160) of four gDNAs should be proposed to

28 be established as the 1st International Genetic Reference Panel for Haemophilia A, Intron 22 Inversion, Human gDNA.

Closing remarks – In his concluding remarks, the chair invited the audience to submit work related to standardization issues relevant to the mandate of this subcommittee for possible presentation in the next meeting. He thanked all the co-chairs, speakers and the audience and closed the meeting at 12:00 noon on the 5th of July, 2008.

Submitted by A. Srivastava

Fibrinogen and Factor XIII

3 July 2008 Vienna, Austria

Chairs: M. de Maat (The Netherlands) and R. Seitz (Germany) Co-Chairs: R. Ariens (UK), A. Inbal (Israel), H. Kohler (Switzerland), J. Koopman (The Netherlands), M. Maurer (USA), L. Medved (USA), M. Neerman-Arbez (Switzerland), J. Weisel (USA)

DRAFT

Factor XIII

Standardisation work

The report of the 1st Business Meeting of FXIII Standardization Working Party in 2008. Prof. A. Ichinose, L. Muszbek, R. Seitz, R. Ariens, S. Raut, and H. Kohler. Prof. Ichinose reported the summary of the 1st Business Meeting of FXIII Standardization Working Party (former FXIII Standardization Working Group) in 2008, chaired by Prof. Akitada Ichinose (A. Ichinose, L. Muszbek, R. Seitz, R. Ariens, S. Raut, and H. Kohler), held in Wiesbaden 4:00 PM-5:30 PM, Feb. 20, 2008, after the Factor XIII Symposium at the GTH meetings. Major agenda were; 1) Merging Issue of two subcommittees, Factor XIII and Fibrinogen (already merged, likely to be a single chair), 2) Chairmanship of the merged subcommittee (a candidate from the Factor XIII side; Prof. Hans Kohler, because of the current co-chair), 3) Standardization Working Party (must continue), 4) Negotiation with the SSC HQ (financial support for SWP, single or double chair?), 5) Negotiation with the Fibrinogen Subcommittee (about joint chair, rotation?). The SWP meeting also confirmed the current condition: 1) Members’ situations; funding, possible conflict of interests, 2) Needed Costs (requested from 2 labs.); ~7000 USD (experiments), ~4000 USD (travel), 3) the SSC policies of 2004 and of 2006-8, 4) Possible supportive companies; A (plasma-derived Concentrates), B (rec XIII-A), etc., 5) Possible other organizations to co- operate; IFCC, (not JSTH, JCT), 6) The choices; dissolution, pause, or continue. Decision to be made in Vienna in July.

L. Muszbek (Hungary) gave an update on methodological problems concerning the measurement of FXIII activity and antigen in FXIII concentrates. When calibrated against company standards, FXIII activity estimates showed wide differences, which diminished with calibration against 1st IS. Antigen values were ca. 9 to 13% higher than activity, though both were calibrated against 1st IS; this might be due to some loss of activity during virus inactivation (pasteurisation). If concentrate was diluted with buffer, the Berichrome test grossly underestimated activity; addition of extra thrombin did not help. In other tests, concentrate FXIII activity was ca. 16% higher with buffer or buffer + fibrinogen than with deficient plasma as diluent, suggesting the possibility of inhibitory constituents in plasma. The prudent choice of the diluent is crucial, dilution with plasma might reflect more closely the physiologic situation, while it may to some extent underestimate the catalytic activity in the concentrate. A concentrate standards appears useful, and needs to be calibrated against the IS. In the discussion, R. Seitz noted that the experience in other cases (e.g. FVIII) showed that dilutuion with deficient plasma did not avoid the need to have a concentrate standard.

S. Raut (UK) presented the current concepts and available materials necessary for further experiments needed, before the actual standardisation can proceed. For the measurement of recombinant FXIII, particular aspects have to be considered; this preparation is of higher purity than the present plasma products, and contains only subunit A. Thus, the presence of subunit B in the system (e.g. dilution with deficient plasma) is an important point. So far, it is open, whether one common or separate plasma derived and recombinant concentrate standards are needed; candidate materials of both types need to be included in the experiments.

Clinical minisymposium

Diagnosis and Management of patients with transient/acquired factor XIII (FXIII) deficiency; Re-revisit.

By Ichinose A1, Souri M1, Sasabe M2, Kotani N2, Mizobe T3, Tanaka A3, Tsukada J3, Ishida F4, Ito T4, Sugita K5, Aki K6, Sawada A7, Higasa S7, Nishikawa T8, Eura R8, Kawakami K8 , Matsuura Y9, Tamai Y10. [1Yamagata Univ., 2Matsuyama Red Cross Hosp., 3Occ. Env. Health Coll., 4Shinsyu Univ., 5Dokkyo Med. Coll., 6Nippon Med. Coll., 7Hyogo Med. Coll., and 8Kagoshima Mun. Hosp., 9Narita Red Cross Hosp., 10Hirosaki Univ. (Japan)] Prof. Ichinose discussed the increasing bleeding disorder due to transient/acquired factor XIII (FXIII) deficiency. He summarized 13 cases of this disease state, among which about a half turned into having inhibitors against FXIII, and the remaining half being idiopathic. He presented several representative cases including 1) a life-threatening cervical bleeding case of 3 y.o. boy, who was saved by the replacement therapy using FXIII concentrates, 2) a case of type I complete inhibitor of FXIII, 3) a case of type II incomplete inhibitor of FXIII, 4) a case with a possible inhibitor against the fibrin cross-liking (ligation) reaction, and 5) a case with a inhibitor against the activation cleavage step by thrombin. He reviewed the current status of ‘Acquired Haemophilia’ mainly due to antibodies against Factor VIII, and proposed to call acquired FXIII deficiency as ‘Acquired Haemophilia XIII’. He also reviewed ‘Hemorrhagic Disorders of Fibrin-stabilization’ published in 1977, and ‘Guidelines of Testing Modalities for Differential Diagnosis of Bleeding Disorders of Fibrin-stabilization’ published in 1994 by Prof. L. Lorand, and pointed out the necessity of rejuvenation, because of emerging additional factors as well as new medicines. Finally, he proposed 1) to conduct a large- scale survey of Acquired Haemophilia XIII, 2) to establish reliable definition for inhibitor and mild FXIII deficiency, 3) to make new recommendation for/guideline on global screening tests of Acquired Haemophilia XIII, 4) to make some recommendation for/guideline on diagnosis & management of Acquired Haemophilia XIII including.

H.P.Kohler (Switzerland) talked about „mild“ reduction of FXIII: between bleeding and thrombosis. Acquired non-immunological reduction of FXIII antigen levels is mainly explained by consumption due to various medical conditions. Such a consumption can lead to a significant decrease in FXIII antigen levels far below the lower normal range limit. However, smaller reduction of FXIII antigen levels within the “normal range” can also be associated with severity of disease or even clinical outcome. Examples from patients with stroke, pulmonary embolism, sepsis and from subjects undergoing surgery give evidence that the so called “normal range” of FXIII antigen levels has possibly to be reconsidered.

Z. Bereczky (Hungary) presented the results of a case control study assessing risk factors in more than 900 patients admitted to the hospital for coronary angiography. Subgroups were formed according the presence of significant (>50%) stenosis of coronary vessels and/or Acute myocardial infarction. Elevated FXIII level turned out to be an independent risk factor in women. Similar results were found also in patients with peripheral arterial disease. Thus, elevated FXIII levels appear to be a gender specific risk factor of atherothrombotic diseases.

É. Katona (Hungary) showed measurements of Factor XIII in body fluids other than plasma. In bronchoalveolar lavage fluid FXIII activity can be found mainly as free subunit A, probably stemming from macrophages as demonstrated by FACS analysis. FXIII levels are elevated in inflammatory bronchoalveolar disorders. In normal cerebrospinal fluid, obtained during lumbar anaesthesia, low FXIII levels are present. Also in tears FXIII can be found. Stimulation of tear flow with intranasal alcohol can strongly increase the volume of tears, but not FXIII concentration.

Fibrinogen

There has been some concerns regarding the availabiltiy, stability and validation of the high fibrinogen standard. In addition, this standard was not approved by the WHO. Therefore, Sanj Raut of the NIBSC explained to us the procedures that the NIBSC used for the standards and he started the discussion on the need for a ± 10 g/l standard. The audience did not see a need for such a standard, they were happy with the currently available standards. It was agreed that at the Fibrinogen Workshop (In Venice in July) Moniek de Maat will ask the participants for their opinion, and decide after that whether a new high fibrinogen standard is required (added 13.07: also at the Fibrinogen Workshop there was no interest expressed in a high fibrinogen standard).

There are a number of fibrinogen assays available and Ann Rumley presented a comparison of three assays (Clauss, PT-derived and nephelometric method) in prediction of cardiovascular risk using the Monica study. Althought the nephelometric method showed a weak correlation with the two functional assays (R±0.6), the

31 relationship of the three assays with risk was comparable. Combination of different assays did not improve the risk prediction.

Leonid Medved and John Weisel present the final version of the fibrinogen/fibrin nomenclature that has been developed over the last years. The manuscript has been prepared and approval of the SSC was asked. The only question that was raised was about the genetic nomenclature: it was decided that both the numbering, based on the mature protein, and the new numbering, based on the advise of the Standardisation Committee Nomenclature Working Group and the HUGO Gene Nomenclature Committee (HGNC), would be used in the manuscript. The manuscript has been circulated to fibrinogen experts and approved by them.

The Fibrinogen Variants Database is growing and Michel Hanss told us that at the moment 454 molecular abnormalities are included, of which 248 in the Aα-chain (82 mutations Aα16), 66 in the Bβ-chain and 137 in the γ-chain. It appeared that a large number of mutations associated with bleeding problems were found in the N-terminus of the Aα-chain, those with amyloidosis were in the C-terminus of the Aα-chain, and those associated with thrombosis were often in the C-terminus of the γ-chain, at positions 14 of the Bβ-chain and 554 of the Aα-chain. Although interesting, these data are only observations depending of the way cases have been collected and described. Several modifications of the data presentation have been proposed, including progressive retranscription of the abnormalities on a different reference sequence, and the translation of mature protein location to native protein location. Direct submission of cases should allow maintaining an accurate representation of variant occurrence.

A new item for standardization is the description of the fibrin clot structure. More and more researchers are measuring the fibrin clot structure characteristics, but there is no consensus on how this is done. Marlien Pieters gave a very complete overview of the methods that are being used at the moment and of their advantages and disadvantages. It was agreed that fibrin clot structure is potentially an interesting variable and that it would be good to have a minimum set of standardized variables available.

Submitted by M. de Maat and R. Seitz

Fibrinolysis

4 July 2008 Vienna, Austria

Chair: Colin Longstaff, UK Co-Chairs: Carl-Erik H. Dempfle, Germany; Ann Gils, Belgium; Dirk Hendriks, Belgium; Osamu Matsuo, Japan; Michael E. Nesheim, Canada

DRAFT

TAFI/CPU Chaired by D Hendriks.

Update on a sensitive assay for functional TAFIa (CPU) in plasma

M Nesheim presented an update on a sensitive assay for functional TAFIa (CPU) in plasma. The assay is based on the ability of TAFIa to release bound fluorescent plasminogen from soluble high molecular weight fibrin degradation products that have covalently attached QSY moieties which quench the fluorescence of the bound plasminogen. When the plasminogen is released the fluorescence intensity increases and the rate of this can be used to measure the level of TAFIa in the sample. The level of TAFIa in a small group of normals was shown to be 20 pM with a range of around 5 to 30 pM. The assay was applied to chimp samples in experiments in which coagulation in vivo was stimulated with a combination of factor Xa and PCPS vesicles infused as a bolus. TAFIa levels at levels as high as 6000 pM were observed within 10 minutes of the infusion. Very high levels (35,000 pM) were found in vivo in lethal E. coli induced sepsis in a baboon model. A modest (2-fold) increase was found in samples of unstable, but not stable, angina in humans. In whole blood in vitro with clotting induced with tissue factor in the presence of and intrinsic pathway inhibitor, TAFIa was found to increase to levels of the order of 2500 pM after clotting, with a time course that tracked very closely to the thrombin-antithrombin complex time course. The extent of TAFIa activation was measured in the same way with 14 hemophiliac samples. The results ranged from values less than one tenth that of normal to near normal. One protein C deficient sample showed supra normal TAFIa generation. These results indicate that readily measured TAFIa levels appear in plasma samples under a variety of conditions and this assay is sensitive enough to measure TAFIa levels down to resting levels which are low

A new substrate allowing sensitive and specific determination of proCPU/TAFI and CPU/TAFIa in plasma samples

E Heylen presented work on a new sensitive assay for CPU (TAFIa) in plasma samples. There is a need for new sensitive assays to determine the importance of CPU levels in the circulation for population studies and during treatments such as thrombolysis for stroke. The assay is based on new sensitive small substrates for CPU which have improved specificity (kcat/Km) over CPN which has a constitutive activity in plasma and complicates the activity determination of CPU. The zymogen form, proCPU (TAFI) also has some intrinsic activity on small synthetic substrates which requires correction. The assay method was described and sensitivity discussed in relation to the expected ranges of CPU in circulation. Further validation of the assay is underway.

Comparison of different methods for measuring human TAFI

Pierre Morange presented an overview and data comparing different methods for measuring human TAFI. Assays may be complicated by the different forms present in circulation, including zymogen TAFI (which may also be complexed with plasminogen), active TAFIa and the conformationally inactive form, TAFIai. There are a number of commercial assays based on activity measurements of TAFI following activation with Thrombin/Thrombomodulin and immunoassays. Some of the immunoassays have different sensitivities for isoforms of TAFI with Ile/Thr at position 325, which in the past has led to overestimation of the genotype effect of this polymorphism. Other polymorphisms have been identified in the gene promoter region that can affect circulating TAFI levels. A number of studies have been performed to investigate correlation of circulating TAFI

33 or TAFIa + TAFIai levels with coronary artery disease and stroke. The prospective PRIME Study evaluated the association between TAFI plasma levels and the risk of coronary disease. in 10,000 healthy men recruited in France and Northern Ireland, TAFI plasma levels were similar in individuals with and without cardiovascular events after 5 years of follow up. However, it important to note that this assay is poorly sensitive to TAFIa and measure mostly the zymogen. Recent data have shown that the amount of activated TAFI (TAFIa) plays a more crucial role than that of TAFI in retarding fibrinolysis. This has led to the development of new TAFI assays measuring either exclusively TAFIa, or associated with TAFIai (TAFIa/TAFIai) or the released TAFI activating peptide (TAFI-AP). It has been recently shown that plasma levels of TAFI-AP (measured by a specific ELISA) were associated with the 4 major types of ischemic stroke in the retrospective SAHLSIS Study. The association between TAFI levels (using 4 TAFI measurements: two evaluating the zymogen, one measuring the TAFIa/TAFIai and the last one the TAFI-AP) and the risk of cardiovascular events have been assessed in the Atherogene Study. This study has included 1668 individuals from Germany with a significant coronary disease. These individuals were followed up for 3 years. TAFIa/TAFIai plasma levels measured at baseline were associated with an increased risk of cardiovascular death. This association was not observed with the test measuring the zymogen or with the ELISA measuring TAFI-AP.

Discussion

D Hendriks presented a short summary and chaired a discussion of the current problems and questions surrounding measurement of TAFI/CPU both zymogen and activated species. Circulating levels of the active enzyme may be of special interest but are very low and few assays have the necessary sensitivity. It is also not clear how to compare results from an assay such as that described by M Nesheim with immuno assays measuring activation peptide, for example. There are also a number of questions and problems surrounding the collection and processing of samples used to measure TAFIa/CPU due to the well known thermal instability at temperatures above zero deg. C. At this stage it was proposed that a collaborative study comparing different assay methods on a common set of samples to measure TAFI/proCPU would be useful and achievable. There was a discussion on the design of such a study including what samples and the number of samples should be used. It would be useful to include samples with different polymorphisms at position 325. Consideration was also given to the preparation of clearly defined samples formed by adding back purified (native or recombinant TAFI/proCPU) to depleted plasma. Further discussion will take place over the coming months to finalise the details of a study before samples can be shipped via NIBSC. The audience were requested to contact the chair and co-chairs of the subcommittee to express an interest in taking part and in making further suggestions.

D-dimer Chaired by C-E Dempfle

Introduction

C-E Dempfle made a short additional presentation to summarise the current situation regarding harmonization approaches for the large number of d-dimer methods currently in use. C-E Dempfle showed results from the Fibrin Assay Comparison Trials (FACT) parts 4 and 5. FACT4 compared assay reactivity of fibrin fragment D- dimer and pooled plasma from patients with DIC, using serial dilutions in plasma from healthy blood donors, and in buffer. A procedure for homogenisation of D-dimer samples was presented which allows quantitation of d-dimer in plasma following immunoblotting. This method has been found to be reproducible but needs testing in more laboratories. A request was put forward for laboratories to contact C-E Dempfle who would provide the method and further help where needed. Data were presented using the harmonisation procedure proposed on 50 plasma samples and a number of assays These results appeared to validate the use of 0.5 ug/ml cut off used in many assays.

Update on the NEQAS EQA for d-dimer

Ian Jennings covered the experience of the UK NEQAS studies on d-dimer determinations and showed how harmonisation procedures could be used successfully to improve inter-laboratory variability. However, between laboratory agreement remains poor in the absence of a suitable international reference preparation for d-dimer in plasma. Further problems were also identified including the variety of units reported and lack of local determination of cut-off values when d-dimer is used for the exclusion of DVT.

Report on the d-dimer harmonisation standardisation project-3 of JSLH for 13 d-dimer tests from 11 companies worldwide.

Optimising the harmonisation strategy to maximise the benefit for clinical practice.

K Fukutake presented results from the Standardization Committee of The Japanese Society of Laboratory Hematology, The Japanese Society of Laboratory Medicine and The Japanese Society of Thrombosis and Hemostasis on Standardization of FDP/D Dimer. These studies have been progressing as a joint project since 2002 in Japan. Project-3 was conducted for optimising the harmonization strategy to maximise the benefit for clinical practice in early 2008 and included 13 dimer tests from 11 companies. Plasmas from 80 patients, whose D Dimer levels were between 5 and 25μg/ml by LPIA, were pooled for calibration material. The pooled plasma was diluted by single normal plasma into 5 levels and samples were preserved at -80Ԩ. Measurement results of original methods were very variable with inter-assay CV around 30% at each dilution. For harmonization, a reference method was chosen and results recalculated using regression curves aligning each method with the reference method. A dramatic improvement in CV to below 8% was noted. Further evaluation of clinical usefulness was established using 16 individual samples from patients, whose D Dimer values were from 5.0 to 25.0 ug. . The harmonization procedure was able improve average CV values from 34% to 9.9%. Benefits and limitations of harmonization and the extent of improvement in variability of d-dimer assays were discussed. It was suggested that a common calibrator of pooled plasma (>80 Patients Pool) is desirable. However, there will always be problems in this area due to the variable molecular composition of the target material and the differing nature of assay antibodies. The limited supply of patient samples may also be a problem for large scale studies.

Fibrinolysis and Plasmin chaired by C Longstaff

Physiological usefulness and limitations of global fibrinolysis measurements

T Urano presented an overview and update on classical tests for global fibrinolysis, concentrating on Euglobulin Clot Lysis Time (ECLT) in a microtitre plate format following absorbance at 340 nm. Preparation of the euglobulin fraction primarily removes alpha-2-antiplasmin and allows the assessment of fibrinolysis. which is dependent on the presence of tPA and PAI-1 and the proportion of free tPA. ECLT correlates with the amount of free tPA or tPA activity and with PAI-1 but not with total tPA antigen. ECLT showed very good positive correlations with total and free-PAI-1. It was found that Ca2+ dramatically shortened ECLT and further investigations showed that this was due to inactivation of PAI-1 by thrombin. Other mechanisms of PAI-1 inactivation were also possible, which involved the contact system. The reproducibility of euglobulin preparation remains a source of variability when using this method.

Update on WHO international standards and SSC secondary standards for tPA antigen and PAI-1 antigen in plasma

C Longstaff provided an update on previous collaborative studies organized through the subcommittee to measure tPA antigen and PAI-1 antigen in plasma. In 2007 results from a collaborate study were reported to the subcommittee showing wide variability of PAI-1 antigen determinations from 12 laboratories using a range of assays. However, the study included 5 samples of PAI-1 pools with a range of PAI-1 and a harmonization procedure demonstrated that conversion of results between methods was possible using an appropriate regression equation. An International Standard (IS) for PAI-1 antigen in plasma would be useful for such a harmonization procedure but this should be calibrated in ng and there is a need to find a way to determine the “real” ng level of PAI-1 in plasma (rather than the apparent ng reported by each method, which are not equivalent).. Data were presented from studies using SPR (Biacore) that may provide a way of estimating the real ng in plasma samples which may provide a route to this calculation. Further work is underway.

Preparation 94/730 was established as a new IS by the ECBS in October 2007 and is now available as the 1st IS for tPA antigen in plasma with a value of 25 ng of tPA. This preparation should be useful in calibrating tPA Elisa assays and replaces a previous preparation that was used for this purpose that is no longer available. The SSC coagulation plasma secondary standard lot 3 was also included in the same collaborative study to calibrate 94/730. When results were reviewed by participants and Fibrinolysis subcommittee members it was also agreed that SSC plasma lot 3 could be assigned a value of 3.0 ng/ml tPA antigen. However, a question

35 was raised about “cryptic tPA” which is not detected by one commercial kit and could have introduced further variability into the study results. This was investigated further it was found that the low values determined by one kit were increased from 1-2 ng/ml up to 5-6 ng/ml if plasma samples were diluted using 2 M lysine. However, this procedure is not routinely performed and it was recommended that the original calibration of 3.0 ng/ml should stand and this has been accepted by the Working Group on Coagulation Standards.

Utility of standards for 4-nitrophenol, 4-methylumbelliferon and 4-nitroaniline for standardizing serine protease activity

C Thelwell summarized recent work at NIBSC on the application of potential standards for 4-nitrophenol and 4- nitroaniline which may be valuable in the determination of molar concentrations of active enzymes by active site titration and for the determination of kcat or katals for active enzyme preparations. Results from 2 previous collaborative studies conducted through NIBSC were reviewed in which enzyme activities or molar concentrations were determined. It appears that a major source of variability in these studies is the preparation of calibration curves for nitrophenol or nitroaniline and/or the collaborators’ spectrophotometers or microtitre plate readers. This source of variability may be improved by having available standard preparations of nitrophenol and nitroaniline. Trial fills have been performed and preparations have been sent out to a number of laboratories to investigate these questions. A request was made for further participants who want to take part in this study. Ultimately it is possible that these preparations will be useful in making new IS (for example plasmin) or in dual labeling existing standards with International Units and moles or katals of activity. Furthermore, active enzymes can be titrated with inhibitors thus calibrating the inhibitor in molar units (as has been done for the 1st IS for Alpha-1-Antitrypsin). These inhibitor preparations may also serve as a route for determining molar concentrations of other enzymes.

Standardising plasmin for therapeutic use

Pete Vandeberg presented work on the standardization of plasmin activity determinations as practiced by Talecris for their commercial product currently in clinical trials. Standardisation is a relevant topic since a number of researchers are investigating the use of plasmin-related proteins for therapeutic applications including thrombolysis but also including ophthalmology and wound healing. Although there is an international standard for Plasmin (97/536), many commercial suppliers of research-grade plasmin use activities derived from different sources, including their own historical assays. This makes comparison of the doses used in published studies difficult. A study was presented examinining the application of active site titration using p- Nitrophenyl-p'-guanidinobenzoate as a titrant to assign a standard values of active mg/mL to a reference preparation of plasmin. This reference preparations, in turn, is used as a standard for a chromogenic potency assay. By using an absolute reference method for reference method assignment, the long-term assignment of potency used for the purpose of dosing can be maintained. In addition, the chromogenic assay using the reference standard is used to compare commercial research grade preparations of plasmin from a variety of different sources. These preparations were found to have a wide range of activities, and a wide range of specific activity, ranging from a low value of 0.07 mg/mg (active plasmin per total protein), up to approximately 1.0 mg/mg for clinical trial grade plasmin produced by Talecris Problems with the historical calibration of the plasmin IS and the withdrawal of the previous NIST standard for 4-nitrophenol suggest there is a need for improved or new standards in this area.

WHO and European activities on standards for plasmin inhibitor (alpha-2-antiplasmin) streptodornase and C1- inhibitor.

C Longstaff closed the session by summarizing progress on other IS and business or relevance to the Subcommittee. A new IS for Streptodornase is under development and should be available in 2009. Previous suggestions for a new standard for plasmin inhibitor (alpha-2-antiplasmin) are currently on hold.

Submitted by C. Longstaff

Haemostasis and Malignancy

4 July 2008 Vienna, Austria

Chair: Martin H. Prins (The Netherlands) Co-Chairs: Giancarlo Agnelli, Italy; Anna Falanga, Italy; Charles W Francis, USA; Agness Y Y Lee, Canada

DRAFT

The following opresentations were held in the meeting, each followed by discussion.

• The BECAT study (prophylaxis of thrombosis in cancer patients carrying a central venous catheter). Dr Ramón Lecumberri, Dr. Eduardo Rocha • The ABEL study (effect of bemiparin on survival of localized non-small cell lung cancer). Dr Ramón Lecumberri, Dr. Eduardo Rocha • The CANBESURE study, prolonged prophylaxis with bemiparin after cancer surgery) Dr Ramón Lecumberri, Dr. Eduardo Rocha • Screening for occult malignancy in patients with idiopathic VTE, the SOME study Ma rc Carrier • Update on INPACT Martin Prins • The Fragmatic trial Fergus MacBeth • The Cancer and thrombosis study Ingrid Pabinger • Recurrent VTE on anticoagulation in patients with malignancy S. Schulman • Predicting recurrent pulmonary embolism or major bleeding in cancer patients with VTE. Manuel Monreal • Standardization of microparticle TF Nigel Key • Update on the development of small molecular weight tissue factor inhibitors for prevention and treatment of thrombosis in malignancy” Fred Rickles • The thrombin generation assay in cancer patients Marina Marchetti • Update on comparison of LMWHs inhibitory effects in the Matrigel assay of angiogenesis Anna Falanga • Working Party on TF standardization in cancer William Pickering / Elaine Gray

There were no important ventures for the SCC to undertake. Many important clinical trials are being undertaken. `the `WP on standardization of TF in cancer is continuing with three sub-projects.

Submitted by M. Prins

Lupus AntiCoagulant/Phospholipiddependent Antibodies

3 July , 2008 Vienna, Austria

Chair: Vittorio Pengo, Italy Co-Chairs: Ph. G. De Groot, The Netherlands; Monica Galli, Italy; Thomas Ortel, USA; Jacob H. Rand, USA; Guido Reber, Switzerland; Armando Tripodi, Italy

During the previous subcommitte in Geneva on July 2007, it was asked to the speakers if guidelines on Lupus Anticoagulant (LAC) diagnosis published in 1995 should be updated. The majority was in favor of a revision.

The working plan was then to divide this issue in four parts: Preanalytical variables in LA determination (G.Reber), Choice of the test(s) (P. de Groot) Procedures (mixing and confirmatory tests) and expression of results (A. Tripodi) Appropriateness of the LA request, interpretation of results (V. Pengo) and to report reccomendations at this meeting for general discussion and suggestions from the audience.

NEW GUIDELINES ON LUPUS ANTICOAGULANT DIAGNOSIS G.Reber: Preanalytical variables in LA determination

Some pre-analytical variables may affect assay results when testing for LA. In the literature there is no data about the influence of sodium citrate concentration (0.109 or 0.129 M) for blood sampling on assay results. However, one study demonstrated that ionized calcium concentration affects the binding of anti-ß2-glycoprotein I dependent lupus anticoagulant antibodies. Another important variable is the amount of residual platelets debris in thawed samples because they can neutralize the antibodies and therefore affect the clotting time of some tests. If LA testing is performed with frozen/thawed samples, it is recommended to centrifuge the fresh sample twice (whole blood and then plasma) and to transfer the supernatant of the second centrifugation in the tube to be frozen. Alternatively, filtration of the plasma with 0.22 µm membranes can be performed. However, for some tests, a single centrifugation above 3500 x g during at least 15 min would suffice.

Freezing the samples should be performed as fast as possible. Therefore, the use of liquid nitrogen is highly recommended. Alternatively, freezing in a acetone/dry ice mixture can be performed. Sample storage prior testing should be made at -80°C and thawing at 37°C in a water bath. Sample content has to be totally immersed.

Unfractioned heparin present in samples has to be neutralized unless the test’ reagent(s) contains a neutralizing agent. Polybrene, anion exchange columns or heparinase can be used. Possible side effects of these agents on assay results of the particular test used should be checked. There are few data in the literature about the effect of LMWH on LA assays. It has to be recalled that the ratio anti-FXa/anti-FIIa activities varies with the brand. It cannot be excluded that samples with high anti-FXa activities from LMWHs with low activity ratio may interfere with some LA assays. Samples from patients receiving anti-vitamin K antagonists should be tested after mixing with normal plasma. For these samples the suitability of a ratio of clotting times obtained with viper venoms insensitive to under-γ-carboxylated prothrombin should be evaluated.

P. de Groot: Choice of the test(s)

Lupus anticoagulant is the assay for the detection of the presence of antiphospholipid antibodies that correlates the best with the presence of thrombosis and fetal loss. Many LA assays have been described. Which tests have the optimal sensitivity and are specific enough?

Most laboratory around the world use dRVVT and aPTT-based aasays which are in fact the most robust tests to detect LA (lower coefficient of variation, ECAT 2008-1)

The subcommittee recommends performing a dRVVT assay due to its high sensitivity and its relative low variability, also when reagents from different suppliers are used. The subcommittee recommends the use of an

APTT as the second test to detect lupus anticoagulant. The APTT, if performed with silica as activator and low phospholipids content, is the second test of choice because it is sensitive for LA and its CV is within acceptable limits. Kaolin as activator is discouraged due to its problematic behaviour in automated coagulometers. Ellagic acid as activator is strongly discouraged due to its insensitivity for lupus anticoagulants. The subcommittee does not recommend the use of a dilute PT due to its variability. The subcommittee does not recommend an Ecarin / Textarin time because there are no commercial assays available based on this principle. The screening of LA is not possible in patient samples with an INR > 1.5. When 1.5 < INR < 3.0, a 1:1 dilution of patient plasma with normal plasma can be considered.

The screening for LA is not possible in patient samples with a thrombin time > 150% of normal plasma. There are commercial dRVVTs that contain an heparin neutralizer and allow measurement of LA up to 0.8 U/mL heparin. Screening for LA in patients on LMWH is possible. Screening for LA in patients on direct thrombin of factor Xa inhibitors is unknown. Aspirin and clopidogrel do not interfere with LA assays.

If prolongation of one of the two assays is > mean + 3SD of 40 healthy controls a mixing assay should be performed.

A Tripodi: Procedures (mixing and confirmatory tests) and expression of results

Normal plasma for mixing studies shall be prepared in house and stored at -70°C according to the following specifications: a) platelet poor (<107/mL) ; b) normal levels of all relevant coagulation factors. A 1:1 proportion of patient to normal plasma without incubation shall be used.

LA is excluded when the coagulation time of mixing test is below the upper limit of reference range. LA can not be determined if the coagulation time of mixing test is longer than the upper limit of the reference range but the Thrombin Time (TT) is also prolonged. Confirmatory test(s) should be performed by increasing the concentration of phospholipids of the screening test(s). Phospholipids concentration should be raised by using phospholipids (either bi-layer or Hexagonal (II) phase. LA is excluded when the coagulation time of the confirmatory test is longer than the upper limit of the reference range. Results should be expressed as ratio of patient to normal pool plasma, ratio of mixing to normal pool plasma and ratio of confirmatory test to normal pool plasma (and their respective normal ranges) The results may be expressed as [(LA screen / LA confirm)pat / (LA screen / LA confirm) control] .

V. Pengo: Appropriateness of the LA request, interpretation of results

Usually vascular thrombosis is related to the presence of aPL when it occurs spontaneously in young subjects without risk factors and pregnancy loss is related to the presence of aPL especially in late pregnancy losses (foetal death).

We could grade the appropriateness of LA request as follows:

Low: VTE or arterial thromboembolism in elderly patients.

Moderate: Accidentally found prolonged aPTT in asymptomatic subjects, early pregnancy loss, provoked VTE in young patients

High: Unprovoked VTE and (unexplained) arterial thrombosis in young patients (<50 years of age), thrombosis in unusual sites, late pregnancy loss, any thombosis or pregnancy morbidity in pts with autoimmune diseases.

Interpretation of results. A positive LA migh be false positive in older patients, if first diagnosed in the lab, if it is appraised as mild and in patients on oral anticoagulant treatment.

Positive LA should be always considered in the context of an antiphospholipid antibody profile comprising at least one solid-phase immunoassay for anticardiolipin (aCL) and anti b2-glycoprotein I (ab2GPI) antibodies. The presence of medium-high titers aCL and ab2GPI antibodies of the same isotype (most often IgG) is in

39 agreement with a positive LA and identify patients at high risk. LA positive only is significantly more frequent in subjects without clinical events or may be false-positive especially if mild in potency.

RELATION BETWEEN LABORATORY FINDINGS AND CLINICAL MANIFESTATIONS P de Groot

In a case-control study it was found that LA is the only test among those exploring antiphospholipid antibodies associated with vascular events (VTE, myocardial infarction and stroke). These results did not change if the cut-off value for LA positivity was increased

Bas de Laat

Validation of assays for the detection of antiphospholipid antibodies has been proven to be a point of concern. We have recently shown that thrombosis-related antibodies are directed against domain I of beta2- glycoprotein I. We have organized an international multicenter study to test whether this finding still holds truth in a large population. 443 patients (all positive for anti-beta2-glycoprotein I antibodies) from 8 clinical centers have been included in this study. We found that, as in the first single center study, anti-domain I antibodies are better correlated with thrombosis (arterial and venous, OR 4.2, 2.7-6.6)) than anti-beta2-glycoprotein I antibodies regardless of domain specificity. To conclude, the anti-domain I antibody ELISA seems to be more specific than the regular anti-beta2-glycoprotein I ELISA and a good addition for the diagnosis of antiphospholipid patients.

J Rand

J. Rand and coworkers reported on current status of the Annexin V resistance assay. Annexin V is an important anticoagulant protein whose properties are a consequence of its forming 2-dimensional crystal shields over phospholipid bilayers containing anionic phospholipid.

Rand presented data describing the Annexin V resistance test, which measures antibody-mediated reduction of Annexin V anticoagulant activity. In blinded studies, the Annexin V resistance assay has been validated as a marker of thrombosis in patients with antiphospholipid syndrome. This test appears to correlate highly with the anti-domain I b2GPI IgG assay and may prove to be a useful tool for identifying patients who are at increased risk for thrombotic events.

ROUND TABLE

During the round table the following issues were discussed:

1. Elimination of Anti Cardiolipin ELISA from tests exploring the presence of antiphospholipid antibodies. 2. Eliminated IgM isotype from ELISA assays. 3. APS diagnosis with a single test performed. 4. All the three tests (LA aCL ab2GPI) performed. 5. Change in APS classification categories.

We had 47 written responses from the audience to these 5 questions.

1. 77% do not want to eliminate anticardiolipin antibody ELISA 2. 50% do not want to eliminate IgM isotype testing in ELISAs. 3. 81% state that APSdiagnosis can not be made when only one of the three tests is performed 4. 70% state that all the three tests (LA aCL ab2GPI) should be performed 5. 62% prefere that APS classification criteria are revised.

Submitted by V. Pengo

Perinatal/Pediatric Hemostasis

3 July 2008 Vienna, Austria

Chair: Gili Kenet, Israel Co-Chairs: Janna M. Journeycake, USA; Prasad Mathew, USA; Paul Monagle, Australia; Wolgang Muntean, Austria; Ulrike Nowak-Gottl, Germany; Nicole Schlegel, France

DRAFT

Thrombosis, risk factors and recurrence Chairs: J Journeycake, USA, U Nowak-Gottl, Germany

1. Meta-analysis of risk factors for recurrent DVT - Ulrike Nowak Gottl, Germany

Recurrence is a major end-point in VTE studies, yet there are few evidence based data about this issue in pediatric patients. The aim of the study was to objectively estimate the impact of inherited thrombophilia (IT) on early onset of venous thromboembolism (VTE) and recurrence in children. A systematic meta-analysis was conducted, yielding data of nearly 3000 pediatric patients and 2000 healthy controls. An association between the IT traits and VTE onset was found. Association with recurrent VTE was found for protein S-, antithrombin- deficiency, factor II variant and combined ITs. Notably, in these studies > 70% of patients had at least one clinical risk factor. This meta-analysis gives evidence that the detection of IT is clinically meaningful in children with VTE and underlines the importance of a pediatric thrombophilia screening program.

Recommendation: Further studies are required to evaluate if risk stratification for anticoagulant therapy, based on underlying thrombophilic traits, will reduce recurrent VTE in pediatric population.

2. FIIG20210A and FVL as risk factors for recurrent thrombosis in children- Guy Young, USA

Factor V Leiden and the FIIG20210A are the most prevalent genetic thrombophilias, however, their role in VTE recurrence in children is unclear. In a multicenter international cohort the rate of VTE recurrence and the time to recurrence in relation to FII, FV and other clinical variables was determined, in consecutively enrolled VTE - patients aged newborn to <18 years carrying the FII variant (n=64) or the FV mutation (n=194). Patients were followed for a median of 58 months. Multivariate analysis showed that presence of the FII mutation, persistence of thrombus at 3-6 months and increasing age were associated with an increased rate of VTE recurrence.

Recommendation: Thrombophilia screening in pediatric VTE patients is recommended, since presence of FIIG20210A may predict higher risk of thrombosis recurrence.

3. CVL thrombosis, results of a multicenter study -Shoshana Revel-Vilk, Israel

Central venous lines (CVL) are an essential part in the treatment of children with cancer. In order to evaluate the risk factors for CVL complications, a multi-center Israeli registry database on children with cancer and CVL was established and 263 children with 415 CVL’s were enrolled and followed for 18 months. Only nine clinical DVT’s evolved, however over 90 CVL obstructions occurred and were associated with line infection.

Recommendation: Uniform guidelines for all pediatric oncology centers will improve the supportive care of children with cancer and are essential for prevention of infections, line occlusions and thrombotic complications.

4. APLS with thrombosis in pediatric patients- Mariana Bonduel, Argentina

This study analyzed the clinical and laboratory manifestations in a pediatric Argentinian APS cohort. APS in children had unique features. LA was the most frequent APLA detected in this pediatric cohort with DVT. Thrombosis related to catheters was frequently detected and may be discussed as a clinical criteria for APS in children. Thrombotic recurrence and death attributable to thrombosis were seen in patients with the co- existence of LA and severe underlying diseases.

Recommendation: Guidelines for definition of pediatric APLA and the management required to decrease thrombosis recurrence in pediatric APS patients should be discussed and validated by further studies. Potential collaboration with Lupus Anticoagulant SSC may be beneficial for future definitions.

Thrombosis complications and therapy. Chairs: G Kenet, Israel, N Schlegel, France

1. Post thrombotic syndrom (PTS) in children - Anjali Sharathkumar , USA

PTS is the long-term complication of pediatric VTE, reported in the literature between 9% and 70%. Clinical characteristics of early and late complications of children with VTEs who were managed by Pediatric coagulation service at University of Michigan (2005-2007) were critically reviewed. Based on the symptoms, an expanded PTS assessment scale (EPTSAS) for children was developed and suggested as tool to assess PTS among pediatric patients.

Recommendation: Clinical PTS scoring system should be applied in further international multicenter pediatric VTE studies. A pediatric SSC working group should try to establish and validate different PTS scores- see also below.

2. PTS-Canadian registry results and discussion: Leonardo bradao, Canada

A retrospective pediatric cohort treated and followed at the Hospital for Sick Children from June 1996 to July 2007 was reviewed for PTS presence following upper arm DVT (UADVT). PTS incidence was approximately 40% with a trend towards increased prevalence and severity in primary UEDVT in children. Long-term follow up showed higher symptom resolution in UEDVT-related PTS, thus its significance deserves further attention.

Recommendation: Potential collaboration with the anti-coagulation control SSC for better definitions of pediatric PTS is suggested and physicians are invited to contact either L Brandao, A Chan or A Sharathkumar for further details.

3. Dosing and monitoring heparin in children during cardiac catheterization - results from a randomized trial (HEARTCAT study). A.Hanslik, C. Male, Vienna, Austria.

Monitoring unfractionated heparin (UFH) in children is problematic because of variable dose-response. A comparison between high dose (100 u/kg) and low dose (50 u/kg) UFH for prevention of thrombosis during elective cardiac catheterization was conducted. Vascular complications were less frequent than anticipated. High and moderately high doses of UFH were well discriminated using aXa,less favourable correlations were noted with application of TG, ACT and APTT.

Recommendation: Further specific and targeted studies of pharmacokinetics, dosing schedules, and monitoring strategies in children should be preformed. Collaborative international data collection will be established by C Male, P Monagle and L Brandao (see below) to evaluate the best monitoring (aXa vs APTT or other) in pediatric patients treated with UFH.

4. Heparin dosing and monitoring in infants- Leonardo Brandao, Canada

The recommended UFH dose for infants is significantly higher than patients > 1 year of age. Data was collected from a cohort of infants < 6 months of age treated with UFH, followed at Thrombosis Service at Sickkids®, Toronto, from 2004-2006. The safety, efficacy and dosing was evaluated. Most infants required

42 higher than recommended UFH dosing in order to achieve therapeutic anti-Xa levels. The incidence of major bleeding was 11%.

Recommendation: Further studies regarding dosing and safety are recommended (see above).

Heparin therapy, Bleeding risk Chairs: W Muntean, Austria, M Rand,Canada

1. Stratification of therapy risk with anticoagulants in neonates- Anthony K Chan, Canada

The safety and efficacy of anticoagulant treatment in neonates with portal vein thrombosis , renal vein thrombosis and cerebral sinus vein thrombosis was presented with about 6% major bleeding and up to 72% efficacy (though high rate of specific late sequels) in previous studies. Multidisciplinary international multicenter studies are suggested in order to stratify age-adjusted therapy according to pre-clinical condition.

Recommendation: International data collection (registry/ further studies) are suggested

2. HIT studies in pediatric patients- Eric Grabowski, USA

Identification of Heparin-associated thrombocytopenia (HIT) in infants and older children is different from that for young and older adults. An upcoming study was presented: comparison of confirmed/suspected HIT cases with controls in order to better understand HIT classification in children. Further studies with emphasize on safety and efficacy doses of newer anticoagulants in small children are encouraged.

Recommendation: Modification of Warkentin criteria and positive ELISA (with adult “cut off” OD values) for pediatric patients is suggested and should be tested by pediatric HIT studies, for further details please contact speaker.

3. Bleeding questionnaire for detection of mild VW in children- W Muntean, Austria

Bleeding questionnaires in order to evaluate risk for bleeding post surgery may not be sensitive enough, since rate of pediatric post surgery bleeding is usually low(1-3 %). A comparison was conducted, using questionnaires, between 88 children in whom a hemorrhagic disorder was ruled out by rigorous laboratory investigation, to a group of 38 children with mild von Willebrand disease (vWD). The detailed questionnaire yielded good results to exclude a hemostatic disorder, but was not a sensitive tool to identify such a disorder.

Recommendation: Use of standardized bleeding questionnaires should be encouraged; however, more sensitive tools for evaluation of bleeding disorders are required. The joint working group of our SSC with the VW-SSC is currently discussing standardization of bleeding questionnaires.

4. New bleeding questionnaire and bleeding risk score- N Schlegel, France

The feasibility and efficacy of the questionnaire and bleeding score in neonates, infants and children who were scheduled to undergo various surgeries was presented. A proposal for use of bleeding score composed of results of bleeding questionnaire, lab evaluation and surgical risk of bleeding was made.

Recommendation: Collaborative international data collection of bleeding score results and outcome is proposed, please contact speaker.

Submitted by G. Kenet

Plasma Coagulation Inhibitors

4 July 2008 Vienna, Austria

Chair: Elaine Gray, UK Co-Chairs: Francesco Bernardi, Italy; Herbert C. Whinna, USA; Steve Kitchen, UK; Richard Marlar, USA; Piet Meijer, The Netherlands

WHO Standards

Replacement of the 2nd International Standard for Antithrombin, Plasma – E Gray

The stock of the current 2nd International Standard for Antithrombin, Plasma is running low and needs to be replaced by the end of 2010. One candidate material will be produced from a pool of at least 30 donations of normal platelet poor plasma from blood transfusion service. As the SSC secondary standard Lot #3 also require a replacement, it is envisaged that the collaborative study will also include the calibration of SSC Lot#4. The collaborative study will be initiated in September 2009 with an ECBS submission date of October 2010.

Report on WHO/ECBS recommendations on the 1st International Standard for Protein C Concentrate – E Gray

In 2006, the WHO/ECBS establishment the 2nd International Standard for Protein C, Plasma. In the same calibration exercise, the SSC secondary standard Lot#3 and a proposed protein C concentrate standard were also assessed. Protein C values were assigned to the SSC secondary standard. For the proposed concentrate standard, all participants agreed with the antigen value, but there was a query over the proposal of assigning a combined chromogenic and clotting functional potency. After further study, the participants and the experts from the Plasma Coagulation Inhibitors subcommittee agreed that the proposed protein C concentrate standard should be value assigned against the 2nd International standard for Protein C Plasma and that the functional potency should be derived from chromogenic assay results only. The ECBS agreed with this proposal and established the 1st International Standard for Protein C, Concentrate in October 2007.

Protein S and Antithrombin

Protein S assays: method-related discrepancies – I Jennings

UK NEQAS thrombophilia screening exercises have revealed marked differences in median results between the three most widely used Protein S activity methods. Results with method X were over a series of exercises an average 17% lower than those with method Z, and 30% lower than method Y. An earlier investigation of protein C activity assay discrepancies had revealed a discrepancy caused by the calibration of a commercial reference plasma. However, with protein S assays, calibration of method X reference plasma did not indicate any calibration issue. Assay of frozen plasma and comparison with free protein S levels showed a greater proportion of results measured lower with method X activity assay than free PS, but the discrepancy was not of the scale seen in the EQA exercises. Lyophilisation of plasma for quality control purposes can introduce artefact with some haemostasis methods, but in house study showed no effect of lyophilisation on the EQA samples employed here. The discrepancy in results therefore remains unexplained at present. Discrepancies in results with different methods will have less clinical impact if locally determined, method specific reference ranges are employed. However, questionnaires from UK NEQAS participants have revealed that 31% employ a locally determined reference range, and 69% of centres using methods x y and z employ literature or manufacturer-sourced reference ranges; approximately 50% of centres report identical lower limits of reference ranges regardless of method employed. Clinical interpretations for EQA samples and potentially clinical samples therefore differ with these different protein S activity kits.

Use of different Antithrombin assays to characterise subjects with antithrombin deficiency – P Cooper

Antithrombin can be measured by functional and antigenic assays, however, antigenic assays may fail to detect subjects with type II antithrombin deficiency and are therefore inappropriate for screening for antithrombin deficiency. The most common functional antithrombin assay is the chromogenic assay, where antithrombin is activated by heparin and then inhibits either thrombin or factor Xa, and residual enzyme is detected when it cleaves a chromogenic substrate. Chromogenic antithrombin assays vary in that either thrombin or factor Xa may be inhibited; heparin and buffer composition also vary as do chromogenic substrate, incubation time and detector settings. Since 1985, it has been known that human thrombin may be significantly inhibited by heparin cofactor II in antithrombin assays with incubation time greater than 30 seconds, resulting in reduced specificity. Assays using bovine thrombin are much less influenced by heparin cofactor II, and factor Xa-based assays are unaffected. Antithrombin level measured with bovine thrombin-based assay is often reduced with antithrombin Cambridge II (Ala384Ser) whereas the level is normal with factor Xa-based assay; this functional deficiency is the most common cause of heritable antithrombin deficiency in the United Kingdom. Sensitivity to type II defects can be increased by measuring antigenic antithrombin level on subjects with borderline antithrombin activity and then comparing this to the activity level, by ratio. Antithrombin Denver (Ser394Leu) is a rarer defect which may be detected by bovine thrombin-based assay, but remains undetected by factor Xa-based assay. Antithrombin Stockholm (Gly392Asp) has also been reported to be detected by thrombin-based assays but not by all factor Xa-based assays. Heparin binding site defects of antithrombin are best detected with short (30 second) incubation of sample with thrombin; short incubation time may also improve detection of HBS defects with factor Xa-based assay, but different manufacturer’s kits have different sensitivities to different HBS defects. In addition to short incubation times increasing sensitivity to HBS defects, short incubation time of sample with bovine thrombin increases sensitivity to a type IIRS defect, antithrombin Sheffield/Glasgow (Arg393His). We conclude that antithrombin assays vary in sensitivity to antithrombin deficiency and one type of assay cannot be guaranteed to detect all type II antithrombin defects. Reducing incubation time of enzyme with test dilution can sometimes increase an assay’s sensitivity to type II defects; therefore a short (30 second) incubation time is recommended. Phenotype and genetic studies are necessary to define type II antithrombin deficiency.

Global Coagulation Haemostatic Tests

Progress on the activities of the Working Party on Thrombin Generation Tests – E Gray

The Working Party agreed that a reference plasma should be established as NIBSC research reagent and made available as soon as possible. The Party also planned to initiate a new study on FVIII inhibitors plasmas and heparinised plasma in the coming year.

Thrombin Generation: Experience of external quality control surveys – P Meijer

The results from the first external quality assessment surveys for thrombin generation was presented. Three different kits with 5 different methods were used by the participants. Different results were obtained for the read-out parameters evaluated and this was due to the differences in test formats. The inter-laboratory variability was found to be greater than 10%. The different methods had varying sensitivity to different levels of haemostasis factors. The different methods were also found to have different sensitivity to microparticles.

Monitoring Thrombin Generation: Do We Need Addition of Corn Trypsin Inhibitor? - H Spronk, A Dielis, R Oerle, J Govers-Riemslag, K Hamulyák, H Ten Cate

Background: Thrombin generation in calibrated automated thrombography (CAT) could in the future potentially be used as a routine method for measurement of coagulability in clinical diagnostics. Although contact activation does not seem to play a role under physiological conditions in vivo, it has been argued in literature that it might influence in vitro measurements of thrombin generation thereby acting as an unpredictable preanalytical variable which produces inaccuracy and imprecision. The aim of the current study was to investigate the influence of contact activation on thrombin generation and the exigency of corn trypsin inhibitor (CTI) to abolish contact activation in thrombin generation measurement at low tissue factor (TF) concentrations. Methods: Thrombin generation was performed by using CAT, thereby determining the endogenous thrombin potential (ETP) and the lag time. The presence of activated factor XII (FXIIa) and activated factor XI (FXIa) was measured using ELISAs. Results: Addition of CTI after plasma preparation had no significant influence on thrombin generation triggered with 0.25 pM TF or higher, as demonstrated by unalterded ETP and lag time

45 values between analysis with and without CTI. Addition of CTI before blood collection reduced the peak height in thrombin generation triggered with 0.5 or 1 pM TF. The ETP and lag time, however, remained unaltered. In contrast, a significant decrease in both ETP and peak height was observed only when the reaction was triggered with 0.5pM TF. Conclusion: This study demonstrated that addition of CTI after plasma separation is not necessary when triggering with TF concentrations of 0.25 pM and higher. Furthermore, it was demonstrated that it is not needed to pre-fill blood collecting tubes with CTI when measuring with TF concentrations of 1pM and higher.

A Protac®-modified Thrombin Generaton Assay to identifyIndividuals at Higher Thrombotic Risk – A Gatt

There is a need for good laboratory prothrombotic markers to identify individuals at high risk of venous thromboembolism. Thrombin generation estimation is an attractive test, however, it is not sensitive enough to changes in the protein C pathway and may lack sensitivity to prothrombotic states. The addition of Protac, a snake venom extract that activates protein C can improve the sensitivity of thrombin generation test. Protac® has s dose dependent inhibitory effect on the endogenous thrombin potential (ETP). Blood from a group of patients with inherited thrombophilia, their first degree family members with no detectable defect and control subjects were tested. ETP and %ETP correlates inversely with protein C, proein S, Activated Protein C Resistence (APCR) and age, whereas there is a positive correlation between the ETP and FVIII, fibrinogen and age and inverse relationship with antithrombin. The Protac-TG assay is sensitive to all the constituents of the PC pathway. The results show why a positive family history of VTE is a risk factor for thrombosis despite negative standard thrombophilia tests. It may also help discriminate between individuals with FVL who have a higher prothrombotic risk than others with the same defect.

Reversible thrombin inhibition in the thrombin generation test - C. Kluft, P. Meijer, R. Kret, R. Laterveer, J. Burggraaf

The direct thrombin inhibitor, hirudin, induces a prolongation of the lag phase in tissue factor driven thrombin generation tests and shows minimal effect on peak thrombin in the therapeutic range. The lag phase effect is consistently observed also for synthetic reversible direct thrombin inhibitors (rDTIs: bivalirudin, argatroban, melagatran, dabigatran) This is in contrast to effects of rDTIs on peak thrombin which are different among test variants and subject to direct effects on the read-out variable, thrombin activity, by the rDTIs and redistribution of thrombin over inhibitors.

An increase in F 1+2 as a marker of prothrombin conversion happens in the therapeutic range of the rDTIs and presents an additional read-out that requires further study. The lag phase is the presently advised robust effect variable. Thrombin inhibitors are used at high concentrations relative to the inhibition constant for thrombin and suspected to exert actions on other coagulation enzymes as well.

We devised two continuous chromogenic Factor Xa generation assays, starting with a PT reagens (Tissue factor) or an APTT reagens (contact activation). In these assays thrombin participation was excluded by addition of an excess hirudin.

Results in the PT-variant revealed a moderate inhibition of factor Xa by melagatran and dabigatran and a more pronounced one by argatroban. Factor Xa generation was inhibited only at higher concentrations of all rDTIs. The concentrations for significant inhibition in the factor Xa generation test and 2 fold prolongation of the standard PT test were similar (2,5 – 5 µM) suggesting that this effect drives the PT inhibition. Results in the APTT-variant showed inhibition of factor Xa generation and of contact factors at lower concentrations ( 0,5 – 1,5 µM) than the PT variant. This was in agreement with the lower levels to achieve 2 fold prolongation in the standard APTT test relative to the PT. It is suggested that the contact activation inhibition of the rDTIs drives the APTT effects. Results with contact activated thrombin generation tests are pending, but this variant is expected to reveal best the main other effects of rDTIs rather than the tissue factor driven variant. It is concluded that rDTIs, by virtue of the use of high dosages exert multiple effects in coagulation of which the effect on the contact system happens already within the therapeutic range.

Monitoring rDTIs can focus on different aspects (antithrombin = lag phase TGT and ecarin clotting time), factor Xa effects (PT) and contact activation effects (APTT). Submitted by E. Gray

Plasma KallikreinKinin System

3 July 2008 Vienna, Austria

Chair: David Gailani, USA Co-Chairs: Keith McCrae, USA; Thomas Renne, Germany; Alvin Schmaier, USA

The meeting of the Plasma Kallikrein-Kinin subcommittee SSC was held Thursday July 3, 2008 in the Schnitzler room from 2-6 p.m. It was well attended with 54 participants (counted), and we may need a larger room for next year's meeting. Lively discussions followed each presentation. The participants discussed changing the name of the subcommittee, and proposed that “plasma contact activation system” would be more appropriate. While this would represent a switch back to the old subcommittee name, the current name does not reflect certain important aspects of our field.

Dr. Thomas Renné reported on the pathophysiology of a new form of hereditary angioedema (HAE III) with normal C1 inhibitor plasma levels and function. Genome wide linkage studies associated HAE III with a point mutation in the gene for coagulation factor XII that results in a substitution at amino acid position 309. The development of a mutation-specific antibody for diagnosis of the disease from patient plasma was discussed. The antibody and screening tests were offered to physicians that take care of HAE patients having normal C1 inhibitor. In the second part of his talk, Dr. Renné showed that PKA-regulated complexes of spectrin and VASP are critical for the regulation of vascular permeability. Defective spectrin/VASP function or binding increases leakage of endothelial cell monolayers, and raises vascular permeability triggered by bradykinin in genetically altered mouse models.

Dr. Niccola Mutch introduced polyphosphates as new contributor to the hemostatic system. Polyphosphates are evolutionary old molecules originally described in simple eukaryotes. Dr. Mutch reviewed the literature on the subject and introduced methods to detect polyphosphates. Polyphosphates are present in human plasma and are concentrated in platelet dense granules. Polyphosphates bind and activate contact system proteins such as factors XII and XI and plasma prekallikrein but not kininogens, and efficiently trigger factor XII- dependent fibrin formation. Their absence may play a role in impaired thrombin formation and bleeding in patients with the Hernansky-Pudlak Syndrome.

Dr. Jonas Emsley showed recent factor XI crystal structure data and discussed its interactions with binding- proteins. The dimeric configuration of factor XI brings domains A1 and A3 together in trans position and is critical for factor XI activation by factor XII or thrombin. In contrast, monomeric factor XI is sufficient for factor IX activation. Factor XI A3 domain mediated factor XI binding to GpIb through the factor XI A3 domain has similar features to binding of vWF-domain A1 to GpIb. Factor XI binding to H-kininogen is mediated primarily by the factor XI A2 domain. Modeling data explains how H-kininogen binding to A2 affects factor XI binding to GpIb, and factor XI activation on platelet surfaces by thrombin and activated factor XII. Dr. Emsley showed preliminary data on the structure of activated factor XI (factor XIa), and introduced a Drosophila-based expression system (DES-system) for expression of contact system proteins in high amounts.

Dr. Craig Thelwell reported on efforts to create an international standard for C1 inhibitor, the serpin used to treat patients with hereditary angioedema. The project was initiated in 2006. Currently there are three plasma derived and one recombinant C1 inhibitor preparations on the market. Dr. Thelwell invited laboratories to participate in generating an international standard for C1 inhibitor. Between twelve and twenty laboratories are required for this effort and anyone who is interested in participating is invited to contact Dr. Thelwell for further details.

Dr. Sandip Kanse spoke about recent research on the factor VII–activating protease (FSAP). FSAP activity has a role in various vascular diseases and a specific single nucleotide polymprhism in the protease (Marburg 1 mutation) is associated with an increased risk for carotid stenosis and acute coronary syndrome. In plasma, FSAP is a weak activator of factor VII but readily activates Pro-UPA. The Marburg-1 mutation reduces FASP enzymatic activity, with little effect on substrate specificity. FSAP is activated by binding to polyanions including heparin, polyIC and polyphosphates. Charge density of the polyanion appears to be a critical determinant of

FASP activation. Dr. Kanse introduced recombinant expression systems for FSAP that were used to localized the heparin binding-site to the EGF3 domain. FSAP modulates PDGF receptor signaling and the protease cleaves several PDGF subtypes with importance for neointimal formation and vascular growth in various tumor models in vivo.

Dr. Christiane Mannhalter talked about the relationship between factor XII genetic variation and risk of thrombosis. In the first part of her presentation, she reviewed the current literature on the 46 C>T polymorphism in the 5´ UTR of the factor XII gene, with factor XII plasma levels varying depending on the genotype (C/C > C/T > T/T). The 46 T/T polymorphism appears to be associated with increased thrombotic risk, but the results of various studies are currently controversial. The world-wide distribution of this polymorphism varies greatly, with a large difference in allelic distribution between Caucasian and Oriental populations. In the second part of her presentation, Dr. Mannhalter discussed a large clinical study from Austria that suggests a U-shaped correlation between plasma factor XII levels and mortality. To clarify controversial results regarding an association between factor XII and thrombotic disease, Dr. Mannhalter proposed a prospective multi-centered study. Interested parties are invited to contact her.

Dr. Johann Heemskerk reported studies from his laboratory on collagen-driven factor XII activation. First, he reviewed the literature on the topic and showed his own data using flow chambers. Various collagens efficiently activate the intrinsic pathway of coagulation. Plasma from mice deficient in factor XII or factor XI is resistant to collagen induced clot formation and platelet activation. Using clotting assays and thrombin generation systems, Dr. Heemskerk showed that collagens bind and activated factor XII, and that plasma prekallikrein and H- kininogen are critical cofactors in this process. In the second part of the presentation, he showed that collagen- driven platelet activation and PS-exposure involves phospholipase-signaling.

Submitted by Thomas Renne on behalf of Dr. David Gailani

Platelet Immunology

4 July Vienna, Austria

Chair: A. Greinacher, Germany Co-Chairs: B. Chong, Australia; Y. Gruel, France; V. Kiefel, Germany; H. Kroll, Germany; T. Warkentin, Canada

DRAFT

Welcome A. Greinacher

Chairman presents the updated agenda, which is agreed by the SSC members. Brief information about the regulations of membership in a SSC; ISTH SSC members: SSC Chair, SSC board, subcommittee chairmen. Subcommittee members: all who show up at the meeting. Active subcommittee members: all who participate actively in the SSC meeting Information and discussion on emphasis on education in SSCs: • Additional day for education every alternate year • In ISTH congress years: split SSC subcommittee meeting in two parts: • 1. Educational overview of selected topics (responsible for program: chairmen) • 2. regular format • Total time for SSC meeting 4 hours. • The platelet immunology SSC will devote 60 min at the 2009 meeting for educational purposes. Topics for 3 lectures, 20 min each, will be selected by the chairmen after receiving the topics of state of the art lectures from the Boston congress organizers. Responsible: A. Greinacher for organizing the program 2009, K. Bauer for forwarding the ISTH 2009 scientific program information to PI SSC chair Publications of the SSC: Subcommittee members and working parties can submit results of their work to the subcommittee chair. First review within the subcommittee. Forward to SSC chair: second review round. The review process is AT LEAST as rigorous as for an original manuscript. If review process is positive: SSC chair forwards to JTH editors. Final decision by the Editors Working group of the SSC on nomenclature of genetic variants: Anne Goodeve, Pieter Reitsma, John McVey: Proposal: use of the Human Genome Variation Society (HGVS): www.hgvs.org; Platelet immunology and ISBT need to be part of this project Dr Cecile Kaplan (France) will join the SSC working group. Responsible: C. Kaplan for contacting the nomenclature group. The vWF and pediatric hemostasis SSC form a working group on standardization of bleeding assessment. Interested members of the platelet immunology SSC are invited to join. Responsible: Dr Rodeghiero who already participates in the working group will be the delegate of the PI SSC.

Autoimmune thrombocytopenia Chairs: B. Chong (Australia), V. Kiefel (Germany)

Update: Activities of the Intercontinental Childhood ITP Study Group (ICIS) P. Imbach: Update on the progress of the ongoing observational trials in ITP. While two registries are closed, the PARC study is ongoing. There is a web site via which patients can be entered anonymously. Controversies: the question was raised, how double entries can be avoided if patients see different hematologists for chronic ITP. Prof Imbach will address this at the next PARC study steering committee meeting. Required action by the PI SSC: motivate members to contribute to the PARC registry. Provide an update at the next SSC meeting. Responsible: A. Greinacher when planning the agenda of the next PI SSC. Standardization of terminology and definitions in ITP: an update F. Rodeghiero: information of the SCC about the activities of the EHA. Final results of the EHA working group on ITP were presented. This working group established a nomenclature for clinical symptoms, duration, diagnostic criteria, treatment aims, and a framework for parameters to be addressed in clinical trials on ITP. Several members of the

ISTH platelet immunology SSC were actively enrolled in this working group. The manuscript on the final consensus is submitted for publication and will be soon available. • Controversies: no controversies • Required action by the PI SSC: none

Alloimmune thrombocytopenia Chairs: Y. Gruel (France) and V. Kiefel (Germany)

Platelet nomenclature and process to assign a new HPA number. Update on the process. P. Metcalf presented by C. Kaplan: This presentation discussed the "rules" by which HPA designation of platelet alloantigens is made. New antigens will only be included only upon approval by the "Platelet Nomenclature Committee." A "w" designation is added after the antigen name if an alloantibody against the antithetical antigen has not been reported. The information is made public at http:/www/ebi/ac/ukipd/hpa. The criteria required include minimum levels of scientific data, verification by other laboratories, with overseeing of the process by the nomenclature committee. Six reference labs are designated. The genetic basis of all HPA designations must have been determined for inclusion. Antigens up to HPA-16w are now reported.

• Chair and secretary of nomenclature committee are the current chairs of; ISBT Working Party on Platelet immunology; ISTH SSC Platelet Immunology • Voting members: • Aster RH Milwaukee, USA • Haas M de Amsterdam, The Netherlands • Kaplan C Paris, France • Kekomäki R Helsinki, Finland • Kiefel V * Rostock, Germany • Newman PJ Milwaukee, USA • Ouwehand WH Cambridge, UK • Santoso S Giessen, Germany • Shibata Y Tokyo, Japan • Smith JW Hamilton, Canada • Non-voting members: • Marsh SGE London, UK • Metcalfe P Potters Bar, UK • * current cochair of the PI SSC

Frequency of antigen incompatibilities in NAIT J. W. Smith: Canadian data on alloantibody frequencies were presented, which are in line with published frequencies in other countries. In couples with a newborn suspected for NAIT, the gene frequency for HPA-1a and -1b are differently distributed as compared to the normal population. For all other HPAs there is no difference in the allele distribution between couples suspected for NAIT and controls. Genotyping for HPAs other than HPA 1a/b will not contribute to identify NAIT. Genotyping should be used to confirm incompatibility in case of NAIT and present antibodies.

• Controversies: Dr Kaplan raised the issue of serologically negative NAIT in which genotyping will still be helpful • Required action for PI SSC: none

Implementing antenatal screening for NAIT, proposal for an European initiative C. Kaplan: Previous research shows that post-natal screening for NAIT is most cost-effective and a recent Norwegian study suggests that routine antenatal screening may be advantageous. Dr Kaplan plans to initiate a collaboration for implementing antenatal screening for NAIT antibodies funded by the EU. Interested laboratories should contact Dr Kaplan.

• Discussion: the ISTH PI SSC supports this initiative from Dr Kaplan. • Required action for the PI SSC: none

What is new in NAIT? Summary of the proposal of the NAIT working group for a new ISTH SSC publication on NAIT. J. Bussel, C. Kaplan, H. Kroll (USA, France, Germany) presented by C. Kaplan: An overview of clinical and laboratory issues in diagnosis of NAIT were presented. The working group on NAIT of the PI SSC feels that the recommendations for diagnosis and treatment of NAIT need to be updated since the last ISTH recommendation on this topic dates from 1991.

• Discussion: There was debate as to whether this topic should be the subject of an SSC publication (a vote was taken, with 7 votes "yes" and 7 votes "no"). The PI SSC chairmen will reevaluate a revised version of the manuscript of the working group which should be a very condensed summary of current recommendations without a broad discussion of uncertain areas.

Drug-dependent thrombocytopenias Chairs: H. Kroll (Germany) and T. Warkentin (Canada)

No contributions in 2008

Heparin-induced thrombocytopenia Chairs: B. Chong (Australia), T. Warkentin (Canada)

When should HIT be suspected after cardiac surgery?

Post-cardiac surgery HIT: Greifswald study S. Selleng, A. Greinacher Preliminary results of a prospective study on HIT in 506 patients post cardiac surgery were presented which confirm the previous French study that HIT occurred exclusively in those patients who received UFH post surgery (2.8%) but not in patients receiving LMWH post surgery. These data suggest that a study should be performed comparing the use of LMWH or other non-UFH anticoagulants for therapeutic dose anticoagulation post cardiac surgery.

• Controversies: Dr Kaplan raised the issue of serologically negative NAIT in which genotyping will still be helpful • Required action for PI SSC: none

Post-cardiac surgery and post vascular surgery HIT: Japanese study. Shigeki Miyata: A large prospective study in Japanese patients post cardiac and vascular surgery shows an incidence of HIT which is at least 1 order of magnitude lower than in Western countries, anti PF4/heparin ELISA did not correlate with risk for thrombosis. ELISAs have the risk to overdiagnose HIT in these patients. The most likely reason is the much shorter use of heparin post cardiac surgery in Japan (max 2 days) as compared to Europe or North America.

• Discussion: is this related to clinical practise or to genetic differences. It was suggested to obtain DNA in this type of studies to allow genome-analysis for the identification of genetic risk factors for HIT • Required action for PI SSC: none

Fondaparinux-associated HIT. T. Warkentin: Three published cases of HIT complicating fondaparinux therapy were reviewed (N Engl J Med 2007; Thromb Haemost 2008; J Thromb Haemost 2008). It was hypothesized that there are two mechanisms by which fondaparinux might produce thrombocytopenia: (a) "delayed-onset HIT" mechanism (in which fondaparinux triggers formation of antibodies that activate platelets without the need for heparin, fondaparinux, or any other sulfated polysaccharide—evidence supporting this mechanism was presented in one of the published cases). (b) "fondaparinux-dependent antibodies" (in which antibodies triggered by heparin or other sulfated polysaccharides lead to thrombocytopenia because pharmacologic concentrations of fondaparinux increase antibody-dependent platelet activation—evidence supporting this mechanism was presented in one of the published cases, in which unfractionated heparin appeared to be the trigger of fondaparinux-dependent HIT antibodies. These observations are consistent with the hypothesis that HIT antibodies are heterogeneous. • Controversies: none\

• Required action for PI SSC: this is a highly relevant topic as in many countries fondaparinux is the only available option for prophylactic dose anticoagulation with a non-heparin anticoagulant. The PI SSC should closely monitor further reports on fondaparinux induced HIT. This may need an PI SSC statement within the next 2 years. Reponsible: Dr Warkentin preparing an update on this topic for the next PI SSC meeting

Assays for HIT

Quantitative interpretation of PF4-dependent EIA's. T. E. Warkentin. Results of a study were presented investigating the predictivity of positive PF4-dependent EIA's for strong- positive testing (>50% serotonin release) in the platelet-serotonin release assay (SRA) of sera referred for diagnostic testing for HIT antibodies. The SRA was shown to yield "dichotomous" results, i.e., the vast majority of sera are either negative or give strong positive serotonin release (>50% release). The study showed that weak-positive EIA results in both a commercial EIA (from GTI Inc.) and in an IgG-specific EIA (in-house EIA, McMaster University), i.e., OD from 0.40-1.00 U, are associated with a low frequency (<5%) of platelet-activating antibodies being present. At the other extreme, an EIA result >2.00 OD units has a much higher frequency (~90%) of indicating presence of platelet-activating antibodies. It is recommended that laboratories report quantitative results of EIA tests, and provide an "interpretative guide" for clinicians. Quantitative interpretation of EIA results is more useful for improving diagnostic specificity than use of IgG- specific EIA's. • Controversies: none • Required action of the PI SSC: If these findings are confirmed, the PI SSC should prepare a recommendation on how to report PF4/heparin antigen assay test results. This is a highly relevant area and needs to be followed at the next ISTH SSC meeting.

New data on EIAs. I. Elalamy: Study on 119 patients: comparison Stago PF4/heparin ELISA and Zymotest HIT test. Shows a high correlation between the two tests. Zymotest has less positive results in patients with a low probability of HIT, while it was positive in all patients with a high probability of HIT.

Combining affinity and avidity. J Amiral: Dr Amiral suggests an approach to maintain specificity of clinically relevant antibodies and to reduce sensitivity for non-relevant antibodies. By affinity purification of antibodies using a column coated with PF4/heparin and sequential elution he found 2 peaks of anti- PF4/heparin antibodies. Peak 1 had a low and peak two had a high affinity to PF4/heparin and showed much stronger activation of platelets. By using chaotropic buffers in the assay, the avidity of antibodies can be included into the test. Stronger chaotropic buffers reduce the binding of low affinity binding antibodies. The question is, whether this can be used for improving the specificity for symptomatic HIT. • Proposal for an ISTH PI SSC study: use sera of prospective trials already performed to assess whether chaotropic buffers can increase specificity of HIT tests for clinical HIT. • PI SSC members who are interested in such a study and who have well characterised sera from prospective studies, which could be tested, should contact the chair of the PI SSC: greinach@uni- greifswald.de

New data on the evaluation of the IgG specific tests Y. Gruel: The algorithm combining the 4T’s and PaGIA (a rapid assay detecting antibodies to H/PF4) and defined by C Pouplard et al (JTH, 2007) has been prospectively evaluated in 198 additional patients. In 175 cases (88%), the risk of HIT was low or intermediate and PaGIA was negative. HIT was excluded in all. PaGIA was positive in 21 patients with a low (n=1), intermediate (n=18) or high risk (n=2) and HIT was then confirmed (SRA and ELISA) in 1, 9, and 1 cases respectively. This experience confirms the excellent negative predictive value of low 4T’s and the usefulness of PaGIA as a rapid screening test in clinical practice. If clinical probability of HIT is low or intermediate and the PaGIA is negative: heparin can be continued. However, in patients with low probability but a clear pos test or in patients with a high probability and a negative PaGIA test, alternative anticoagulation should be initiated and the other tests employed such as a PF4/heparin ELISA and a functional assay.

• Controversies: none • Required action of the PI SSC: at the 2009 meeting the PI SSC has to decide whether the available data on the combined use of a clinical scoring system and PF4/heparin antigen assays

are clear enough for allowing the recommendation of a diagnostic algorithm in patients with suspected HIT. • Responsible: Dr Gruel (PI SSC cochair) will update the PI SSC in 2009

Combined interpretation of antigen tests and the HIT score. Y. Gruel: One IgG-specific assay (Zymutest HIA-IgG) has been evaluated in a cohort of 101 patients suspected of HIT. The specificity of this assay was higher (90%) than those of the 2 « global » assays tested (50.8% for the GTI assay and 77% for the Zymutest IgG/A/M) without any loss of sensitivity (97.5%).

Improving the specificity by using the high heparin approach in antigen tests. T. Ortel A retrospective study was reported assessing the usefulness of adding high heparin to samples who test positive in the PF4/heparin ELISA. The study was performed with the GTI test. First the samples of the year 2005 were retrospectively assessed to develop an algorithm and to define the parameters to be assessed in a multivariate analysis. This model was then applied to the samples tested in 2006. The proportion of patients with HIT in the group who showed inhibition of reactivity with high heparin was much higher than in the group of patients who did not show inhibition. However, if the OD in the ELISA is >1.0, there are HIT patients who do not show inhibition by high heparin concentrations despite having HIT. Conclusion: high heparin inhibition of positive sera improves the specificity of the PF4/heparin ELISA for clinically relevant antibodies. However, interpretation of ELISA results with an OD >1.0 require careful interpretation. This approach is currently evaluated prospectively. • Controversies: none

Validation of commercial HIT assays pro and con?

Mechanisms of regulatory control - Requirements for reference material. The perspective of the PEI. R. Seitz: Dr Seitz informed the PI SSC about the procedure and requirements for establishing a reference material for PF4/heparin antibodies. The establishment of WHO International Biological Reference Preparations involves a number of steps summarized as follows: • The need for an International Biological Reference Preparation is recognized by scientific and medical community worldwide. • On the basis of this a case is formally made by the WHO Secretariat to the ECBS. • Working groups of experts set the priorities and characteristics for selection of the candidate reference preparations. • An international collaborative study is carried out before any candidate reference preparation may be considered for establishment by the WHO ECBS. • An internationally agreed unit is attributed to the first International Reference Preparation for biological activity characterization. The continuity of such unit is ensured by replacement with a new batch of reference preparation which must be calibrated against the first or previous reference preparation. • In all cases such studies must show that the proposed materials are suitable to serve as a WHO International Biological Reference Preparations in terms of stability and reliability of the materials. • The Biological Standardization (BS) document which reports the international multi-method collaborative study is peer-reviewed by group of experts and international scientific forums before being submitted to the WHO ECBS. Discussion: the PI SSC feels that there is a big demand for a standard. However, there are important issues to be clarified before the process of establishing a standard can be started. Ideally the material should be from patients with clinical HIT. This is hardly feasible considering the severity of the disease and the transience of the antibodies. Pooling of antibodies from several patients might be problematic as this pool does not reflect the biological diversity of antibodies and might therefore be not able to really test whether a test system detects antibodies in individual patients. The issue of informed consent of the patient and of financing sample collection needs to be clarified. Responsibility: the PI SCC chairmen will work on this topic and will prepare a suggestion at the Boston 2009 SSC meeting.

Vienna. July 2008-07-05 Submitted by A. Greinacher

Platelet Physiology

4 July 4 Vienna, Austria

Chair: Marco Cattaneo, Italy Co-Chairs: Joel S. Bennett, USA; Paul Harrison, UK; Catherine P. M. Hayward, Canada; Dermot Kenny, Republic of Ireland; Alan D. Michelson, USA; Diane Nugent, USA; Steve Watson, UK

The session was mostly focused on the reports from the Working Party on Platelet Aggregation, which formed in July 2006, during the chairmanship of Alan D. Michelson.

Marco Cattaneo, Chairman of the Working Party, briefly outlined the aim and the activities of the Working Party. Aim of the Working Party is to achieve a consensus on recommendations on how to perform light transmission aggregometry in clinical and research laboratories. The first step was to organize a worldwide survey on how light transmission aggregometry is performed in real life. To this end, the Working Party prepared a questionnaire of 129 questions, with the help of experts in the field. The questionnaire was submitted online in February 2007 to ISTH members who expressed their interest to participate, and also to members of External Quality Assurance Organizations (e.g., EQAT, NASCOLA, CISMEL, NEQAS-BC and others). Analysis of the questionnaire was completed in June 2008. Catherine P.M. Hayward reported on the main results of such analysis. In total, 244 clinical laboratories and 115 research laboratories from all over the World, but more frequently from Europe (45%) and North America (34%), responded to the questionnaire. It was quite evident from the results of the survey that there is an extreme heterogeneity in the methodology that the responding laboratories follow to perform light transmission aggregometry. This finding confirms that there is an urgent need for the standardization of the technique.

The second step in the activity of the Working Party aimed at achieving a consensus on how light transmission aggregometry should be performed. In consideration of the lack of published scientific evidence, it was decided to try and reach the consensus using the RAND methodology. The RAND methodology is intended to obtain a formal consensus among expert groups about the appropriateness of health care interventions, particularly when scientific evidence is absent, scarce and/or heterogeneous. Technically, for each clinical scenario, a form is prepared, where each member of a group of experts scores appropriateness from 1 (completely inappropriate) to 9 (fully appropriate). Ballots are blinded to the other members. The two extreme scores (highest and lowest of the experts) are discarded, and the area containing the majority of the ballots defines the classification of the intervention (inappropriate, uncertain, appropriate). Eleven experts were chosen among the current Co-chairs of the subcommittee, the past Co-chairs who served during the chairmanship of Alan D. Michelson (2005-2007) and those who had experience on LTA standardization in other organizations. Forty- eight RAND forms, each containing a statement on how LTA should be performed, were prepared and submitted to each expert. A second run of RAND forms was done a few months later, after modification of some statements, based on the comments and suggestions of the experts during the first run. Presentation of the consensus reached among experts during the first two RAND runs at the meeting of the SSC Subcommittee on Platelet Physiology in Vienna is considered part of the long process to reach a final consensus, because all the comments and criticisms that emerged during the discussion of the consensus will be useful to improve the quality of the consensus.

The last part of the session included two presentations on other platelet function tests.

Paul Harrison analysed most commonly used tests, such as the PFA-100 device, the Verfy Now device and the Multiplate whole blood aggregometer. Each of these tests uses whole blood and therefore avoids most of the issues involved in blood processing for light transmission aggregometry. With the PFA-100 he presented a summary of the literature post our previous SSC document on this device published in JTH in 2006. There are now more than 500 papers published on this device and 200 more since 2006. The consensus statements on the PFA-100 still hold as a screening device. Paul Harrison also presented that there is a new PFA-100 P2Y cartridge in development which may have the potential for measuring responsiveness to clopidogrel and related drugs. He also presented an overview of the meta-analyses of the PFA-100 CEPI cartridge that have been published recently suggesting that the test is predictive of MACE in high risk individuals on aspirin treatment.

He pointed out that this is clearly not true aspirin resistance and that is likely to be a measure of platelet reactivity and is impacted by VWF levels. He then proceeded to discuss platelet reactivity and shortened CT’s measured by the aspirin independent CADP cartridge. There is a growing literature on this latter topic and he also presented some unpublished data from large studies within Oxford (>600) and UMASS (>500). These studies demonstrated contrasting data on the predictive value of shortened CADP CT’s with >100 MACE in each. A meta-analysis of shortened CADP CT’s should be feasible soon. With the VerifyNow device he presented an overview of the 3 cartridges each designed to test the major classes of anti-platelet drugs and highlighted some of the significant recent literature on each. There is increasing evidence that each test can predict poor outcomes in small studies and he discussed some of the large trials that are either planned or in progress, which will determine whether there is clinical benefit in identifying poor responders and adjusting their anti-platelet therapy. It should be feasible to perform a meta-analysis of each test in the future. Lastly he presented an overview of the new Multiplate whole blood aggregometry system which is increasingly popular in Europe and the UK. There is a growing literature beginning to show a number of applications of this technology particularly in measuring drug responsiveness and identifying platelet defects. However, the test is relatively new and more trials are required.

Finally, Larry Frelinger updated the group on less commonly used and newly developed tests of platelet function: Vasodilator-Stimulated Phosphoprotein (VASP), TEG® PlateletMapping™ System, IMPACT® Cone and Plate(let) Analyzer, PlaCor PRT7000™, ThromboVision T-Guide®, Serum TXB2, Urinary 11-dehydro TXB2. Dr. Frelinger’s presentation could be summarized as follows: 1) Platelet function as determined by urinary 11dhTXB2 predicts adverse events in selected populations of aspirin-treated patients, 2) Platelet function as determined by VASP and TEG PlateletMapping predict adverse events in selected populations of clopidogrel-treated patients, 3) VASP assay may be useful to guide therapy, but larger studies are needed, 4) More studies are needed to determine the clinical utility of new point-of-care assays: Impact, ThromboVision T- Guide, PlaCor PRT7000.

Submitted by M. Cattaneo

Predictive Variables and Cardiovascular Disease

4 July 4 Vienna, Austria

Chair: Gordon Lowe, UK Co-Chairs: James Douketis, Canada; Veikko Salomaa, Finland; Alberto Tosetto, Italy

DRAFT

G. Lowe (UK) presented the final Subcommittee report recommendations on associations of haemostatic variables with risk of arterial thrombotic events. This report would now be submitted to the SSC.

J. Douketis (Canada) presented an overview of recurrent venous thromboembolism, emphasizing the need to identify clinical and laboratory predictors of recurrence, which might be used to facilitate clinical decisions on long-term anticoagulant prophylaxis. F. Rosendaal (NL) reviewed clinical predictors, which included male sex, idiopathic presentation, and cancer. These appeared stronger predictors than thrombophilias, although further work on genetic predictors was required. P. Clark (UK) discussed the associations of ABO blood group non-O with thrombosis, including potential mechanisms such as higher levels of the FVIII:VWF complex, and the need for further studies including recurrent thrombosis. O. Wu (UK) presented a systematic review of thrombophilias and recurrent thrombosis. FV Leiden showed a weak association; while further data was required to assess the association of other thrombophilias. J. Douketis (Canada) presented a collaborative meta-analysis of fibrin D-dimer in 7 studies: persistently raised levels after discontinuation of anticoagulants was associated with a 2-3 fold increased risk of recurrence. A. Tosetto (Italy) presented the study design of an ongoing collaborative individual patient meta-analysis of these studies. Results of this subcommittee activity would be presented in Boston in 2009. Possible future subcommittee activities discussed might include collaborations on genetic studies; risk of recurrence in less common thrombophilias; and thrombotic risk factors for recurrent arterial thrombosis.

P. Reitsma (NL) reviewed the genetic architecture of venous thrombosis, as revealed by different types of ongoing studies. A. Carter (UK) reviewed genetic determinants of fibrin structure and function in relation to stroke, including results from the EUROCLOT study. L. Iacoviello (Italy) reviewed the development of studies of genetic associations of coronary heart disease. It was agreed that much further work was required to identify new genetic predictors of thrombotic events, and that it would be useful to have a presentation on new statistical approaches to prediction in Boston in 2009.

Submitted by G. Lowe

Vascular Biology

5 July 2008 Vienna, Austria

Chair: Jean-Marie Freyssinet (France) Co-Chairs: Michael Berndt (Australia), Françoise Dignat-George (France), John Griffin (USA), Peter Newman (USA)

DRAFT

The session was divided into three parts, addressing key issues in vascular biology and related disorders. None of the three topics does specifically belong to the scope of ISTH, which emphasizes the efforts to be made to maintain a leadership in the field and to attract leading speakers.

Receptor shedding and shed receptors as clinical biomarkers of vascular disease was the first topic. The fate of some platelet receptors, and cognate ligand in one case, was reported.

Compromised ITAM-based platelet receptor function ITAM was evidenced in one patient with immune thrombocypenic purpura (ITP). In ITP patients, platelet autoantibodies can impair platelet function, and ITAM receptor dysfunction may account for bleeding tendency (M. Berndt).

Soluble P-selectin has been found a novel independent clinical biomarker for venous thromboembolism in cancer patients. Main tumor origins were breast, lung, gastrointestinal tract, pancreas, kidney, prostate, brain, haematologic malignancies, and others (I. Pabinger).

This underscores the significance of the platelet “sheddome” in pathology. Not only the shed receptors have to be considered by themselves but also the pathways associated with, or accounting for, the shedding process. For instance, P-selectin glycoprotein ligand-1 (PSGL-1) has been reported to regulate P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets. Amazingly, psgl-1 knockout in mice leads to increased agressivity, probably as a neurological consequence of stroke occurring in the absence of proper regulation of soluble P-selectin (D. Wagner). Receptor shedding and shed receptors as clinical biomarkers is considered an appropriate topic to be proposed by this Subcommittee for SSC Educational Sessions planned for the Boston meeting.

Circulating endothelial cells (CEC) are believed to reflect endothelium damage or degeneration whereas endothelial progenitor cells (EPC) are viewed with regenerative potential, at least in cardiovascular disorders, knowing they can also be mobilized in tumor development. Hence, the second session was dedicated to the analysis of circulating cells of endothelial origin detectable in peripheral blood .Technical challenges of standardization relies both on their scarcity and on the overlapping phenotype between EPC and mature CEC.

M. Strijbos addressed the specificity of a single plateform flow cytometry (FCM) method that identified CEC in whole blood as CD34+/ CD45-/ CD31+/CD146+ events. Using DNA analysis and cell sorting, he demonstrated that these cells are in fact “giant platelets” that unspecifically bind antibody to CD146. Such interference may in fact participate in the huge discrepancy between CEC counts reported in the literature using standard FCM (few CEC per µl) or assays based on cell enrichment of CD146+ cells (few CEC per ml).

L. Terstappen (USA) presented an automated instrumentation for CEC analysis that combines isolation of CD146+ cells using paramagnetic nano-particles and image analysis. Criteria for CEC identification included size, morphological properties, presence of nucleus, expression of endothelial markers such as CD105 and negativity for CD45. After control of vene-puncture artefacts, normal values of a few CEC per ml, comparable to those reported in the consensus protocol ( Woywodt, J Thromb Haemost, 2006) were observed while elevated levels were found in cancer patients.

Moving on to EPC, G. Uzan stressed the interest of immuno-depleting lineage positive cells previous to multicolor FCM. He showed that the numbers of Lin-/ CD133+/ CD34+/ KDR+/ 7AAD- cells correlate with those of late EPC outgrowth colonies in normal subjects and in patients with leukaemia or systemic sclerosis.

L. Heymans (Belgium) critically compared 6 different FCM protocols for EPC analysis based on co-expression of CD34 and KDR. Besides recalling technical recommendations applied to rare cell analysis, she showed that, despite a higher potential loss of cells, previous mononuclear cell enrichment by density gradient centrifugation significantly improved counting data reproducibility as compared to whole blood techniques.

M. Pirro (Italy) added to the same technical considerations the interest of measuring both EPC and EPC– derived microparticles (MPs). The presence of KDR+ MP in the circulation was proposed as a marker of apoptosis-related reduction in EPC and was shown to correlate with Framingham score in patients with vascular disorders . Therefore combinations of EPC and EMP levels could be useful to improve the assessment of cardiovascular risk.

This topic has to be developed if ISTH wants to maintain a certain leadership in the field of vascular biology, actively investigated in other specialities, not only cardiovascular biology but cancer biology. The benevolent participation of established investigators, not serving the ISTH, is certainly an encouragement in this sense.

Regarding microparticles (MPs), several years ago, B. Furie emphasized by that owing to the physical laws of light scattering, flow cytometry may not be fully appropriate for enumeration of the sole forward scatter basis. For instance, impedance flow cytometry used to detect MPs in cancer patients does not yield the same figures than flow cytometry, in patients and control subjects. The characteristics of new instruments were reviewed. However the size detection threshold remains ~ 0.4 - 0.5 µm (B. Furie).

Hence, it is clear that MPs enumerated by flow cytometry may just represent a tiny part with respect to the pool of circulating MPs. P. Harrison presented evidence that dynamic light scattering (DLS) can yield valuable information on the size distribution of MPs, showing that only a small percentage of the MP “iceberg” can be seen by conventional flow cytometry since the main part of MPs has a size ≤ 0.4 µm. Furthermore, the size of MPs could vary according the stimulus at the origin of MPs, the smaller ones seem to be associated with pathological conditions (J.-M. Freyssinet). Although a sophisticated technique, DLS is operational in platelet- free plasma and revealed that aggregated lipid particles can be confounded with MPs, which may well occur in hyperlipemic patients. Another point was the discrepancy between the protein or lipid content of MPs and their numbers reported in the literature. In order to better define internal standards, it would therefore be advisable to express MP concentrations in protein and/or lipid equivalents, especially in experiments aimed at exploring MP associated functions.

Thrombin generation can be a valuable tool for assessing the procoagulant potential of MPs (B. Binder). Red blood cell-derived MPs were reported having a higher thrombogenic potential probably owing to their phospholipid composition determined by lipidomics. Again, the recurrent problem is that of a reliable internal standard.

Among MPs circulating in the vascular compartment, those of endothelial origin (EMPs) have to be better defined, which has led to the re-evaluation of the criteria (R. Nieuwland). MPs harbouring the CD62E/CD144/CD146 phenotypes are the more likely to be of endothelial nature and can therefore be considered true EMPs.

The confusing situation with respect to the enumeration of MPs by flow cytometry has led Françoise Dignat- George and Nigel Key to propose a workshop on standardization by using calibrant microbeads that will be distributed free of charge by the manufacturer, in agreement with the conflict-of-interest guidelines provided by the ISTH Executive Director and SSC Chair. Calibrated microbeads should enable to standardize instrument settings, then biological samples could be distributed by the core lab (F. Dignat-George) provided shipment issues are solved with respect to specific national security regulations. Regarding the latter point, Elaine Gray who is used to biological sample exchanges at an international level, has kindly offered help. This action has been presented in the DIC and Malignancy Subcommittee sessions and has raised a real interest as 15 to 20 groups have declared being willing to participate in the workshop. The slides detailing the main aims, features

58 and milestones of this workshop, as well as the form of intent to participate will be available soon for download from the ISTH website.

In summary, in its 2nd year of existence, the Scientific Subcommittee on Vascular Biology has identified three main topics. Two of them, “CEC & EPC“ and “Microparticles“, have to be further investigated at methodological and standardization levels while “Shed Receptors” will be developed in future SSC educational sessions.

Submitted by J-M Freyssinet

Von Willebrand Factor

3-4 July 2008 Vienna, Austria

Chairperson: David Lillicrap (Canada) Co-Chairs: Thomas Abshire (USA), Giancarlo Castaman (Italy), Jorge DiPaola (USA), Jeroen Eikenboom (Netherlands), Emmanuel Favaloro (Australia), Anne Goodeve (UK), Bernhard Lammle (Switzerland), Reinhard Schneppenheim (Germany)

VWF Assays for VWD Diagnosis:

Multimer Standardization: Ulrich Budde (Germany)

• 4 population studies used for standardization purposes. Many methodologic variables. • No significant effects of lyophilization of samples – multimers preserved. • CVs vary significantly between methods especially for the type 2 variants. • Multimer patterns are sometimes mutation-specific. • Medium resolution gels are recommended as “universal” gels for multimer profiles. • Useful to have a supra-normal multimer control.

Collagen Binding: Emmanuel Favaloro (Australia)

• Still major problems with the VWF:RCo assay. ~40% CV in many EQA settings. • VWF:CB improves diagnostic precision. Also adds a complementary functional assay. • Current VWF:CB assays too good for binding VWF. Often, just type 3 collagen – this may be too sensitive. • Need better optimized type I/III VWF:CB assays. Better plasma and concentrate standards also required. • No point in further field testing until we have a more optimized assay.

Alternative Functional Assays: Bob Montgomery (USA) and Hans Deckmyn (Belgium)

• The current aggregometry-based VWF:RCo assay is poorly reproducible and has a poor sensitivity <10%. • We need a better functional platelet-dependent assay. • Instead, Deckmyn assay uses soluble wild type GPIb in an ELISA protocol. Still needs ristocetin to promote binding. Avoids use of human platelet variability. • In the Deckmyn assay – better CV and much better sensitivity (0.0005 U/ml). Correlates with VWF:RCo results. • Needs production of recombinant soluble GPIb. • Potential for using glycocalicin as the soluble GPIb fragment. Need to “re-set” glycocalicin. Good CV <10% sensitivity 0.0005 U/ml. Correlates with soluble GPIb and VWF:RCo assays. • In Montgomery assay – PT-VWD mutants used (enhanced VWF binding) – expressed in HEK293 cells with GPIbbeta and GPIX. Using the GPIb expressing 293 cells, good correlation with VWF:RCo results. No requirement for ristocetin in this assay. Multimer size sensitive. Enhanced by microtiter plate vortexing.

Flow-regulated Assays: John Heemskirk (Netherlands)

• Need to take into account the key role of flow in mediating VWF-GPIb interaction. Recent publication in JTH by Biorheology SSC. • PFA assay one example of a flow-mediated assay. But there are some limitations. • Cone and plate analyzer also an alternative but again with limitations.

• Potential of establishing a plasma bank of abnormal functionality for assessment in flow systems.

VWF Propeptide Assays: Bob Montgomery (USA) and Bas De Laat (Netherlands)

• VWF:pp is a marker of endothelial perturbation. Can also be used as an indirect marker of VWF clearance. Half-lives – VWF:pp 2.5 hrs: VWF:Ag 12 hrs. • Subset of type 1 VWD patients with increased VWF clearance. Higher VWFpp/VWF:Ag ratio. • Sanquin and GTI commercial test comparisons show initial very good correlation of the two VWF:pp assays. • Measurement of molar units or IUs. • Need for a VWF:pp standard. Possibility of ISTH #3 as the standard – assigned as 1.06 U/mL. Assume a VWF:pp to VWF:Ag ratio of 1. • Need to finalize, agree upon, an abbreviation for the propeptide. ?VWFp

Acquired VWD: Ulrich Budde (Germany)

• A rare disorder but may be under-diagnosed. • Disease associations – lymphoproliferative disease, myeloproliferative disease, cardiovascular disease. • Bleeding tendency is high. • Some functional defects measured by especially VWF:CB. • In cardiovascular associated cases often loss of HMW multimers. • Testing for the anti-VWF autoantibodies is problematic. High background binding. • Web site available for acquired VWD registry.

VWF Antibody Testing: David Lillicrap (Canada)

• A rare complication of a rare disease (type 3 VWD). Prevalence ~5%. Incidence unclear. Not in any other form of VWD. • Predictors of antibodies. VWF genotype – deletions and nonsense mutations. • No standardized methodology. Both binding and functional (FVIII binding/platelet binding) inhibitory assays. • Sometimes associated with anaphylactic responses or delayed hypersensitivity. • Therapy possible with rFVIII and/or platelets. • Formalized antibody testing program being established for introduction of rVWF.

Standardized Bleeding Scores:

Adult bleeding scores: Francesco Rodeghiero (Italy), Alberto Tossetto (Italy)

• Different aims in different cases. May be a challenge to have a single score. Should we aim for a universal scoring system or should this be disease-specific? • Main aim is to develop the tool for mild bleeding disorders. • Development of a structured questionnaire. This should be standardized. Should have good inter- observer reproducibility. A score should be developed. Then validate the scoring system prospectively in patients vs controls. • Presentation of some example pages from the Rockefeller on line bleeding database (Barry Coller). • Differentiation of severity in severe bleeding disorders may be difficult due to early score “saturation”.

Pediatric bleeding scores: Paula James (Canada)

• Special issues for children with bleeding. Less chance of hemostatic challenges. Different patterns of bleeding. • Previous studies in children have been semi-quantitative in type.

• Discussion of Pediatric Vicenza Bleeding Questionnaire (-1 to +4). • Three study groups evaluated: normal children (positive score>2), most frequent symptoms minor wounds; prospectively assessed outpatient children (135 subjects) – some mild type 1 VWD cases. The pediatric scoring system showed a very good ROC performance (0.86): final group – children with previous diagnosis of a bleeding disease (average scores of >6).

Preoperative bleeding scores: Christoph Bidlingmaier (Germany)

• Pre-operative bleeding assessment. Large country-wide survey. 555 bleeding reports. 8% bleeding complications in tonsillectomy cases. 2% bleeding in adenoidectomy. • Multitude of ways to screen – assays (PT/PTT etc) and many different questionnaires. • In prospective study 42% +ve bleeding history but normal PTT. 30% +ve bleeding history with a defined bleeding disorder. • A normal bleeding history still missed 10% of bleeding problems. • History problems – language, memory, ethnic contexts. • Now, prospectively using the Pediatric Vicenza scoring system pre-operatively.

ITP bleeding scores: Cindy Neunert (USA)

• Rationale for disease-specific scoring system. Example of ITP vs marrow failure-related thrombocytopenia. • Discussion of prior ITP scoring systems. Some only semi-quantitative but some much more quantitative. • Inverse correlation between platelet count and incidence of bleeding. Correlation between skin and oral bleeding. • Prior studies have not been designed with vigorous methodology. • For future studies – more rigorous methodology required. Validity, reliability and responsiveness should all be included in future plans. Planned to incorporate into the SSC Working Party.

VWF Plasma and Concentrate Standards: Tony Hubbard (UK)

• Currently, 5th International Plasma VWF/FVIII Standard and 1st International VWF/FVIII Concentrate Standard • Both will need to be replaced in next 2 years. • For Plasma Standard already 20,500 ampoules filled. Looking for endorsement by SSC in Boston 2009. • Concentrate standard ampoules being used - x 400-600/year. • Concentrate standard will need endorsement by SSC in May 2010. • Still problems with collagen binding assay for the VWF/FVIII concentrate.

Concentrate functional testing: Andreas Hillarp (Sweden)

• Consideration of automated ristocetin cofactor assays for VWF concentrates. • Different reagents and different equipment. • With the automated testing the RiCof levels of concentrates are all low (~50%). This improves with increasing ristocetin concentration in the assay (up to 1.8 mg/ml). Beware ristocetin precipitation.

Classification of VWD: Javier Batlle (Spain) and David Lillicrap (Canada)

• Should types 2A and 2M be differentiated? • Some variants have been variably classified. • Multimer abnormalities are often very subtle. • There have been rare cases of distinct collagen binding defects (ie. 2M collagen type).. • Differentiation between severe type 1 and type 3 VWD remains problematic.

• Practical advantages of type 2M differentiation?

ADAMTS13

ADAMTS13 Polymorphisms and Mutations in Japan: Koichi Kakame (Japan)

• 11 patients with Upshaw-Schulman Syndrome. R193W most common ADAMTS13 mutation. • Now more then 80 ADAMTS13 mutations worldwide. No hotspots - throughout ADAMTS13 gene. • Six ADAMTS13 polymorphisms prevalent in Japanese. • ADAMTS13 actvity levels lower in males. • Q448E and P475S polymorphisms both have an influence on the ADAMTS13 levels. • Subgroup (1%) of general population with low ADAMTS13 levels – sequenced ADAMTS13 gene. In this group there are some new, not previously-described ADAMTS13 mutations. • Calculation of frequency of inherited ADAMTS13 deficiency – I in 1.14 million (80-150 USS patients in Japan). We do not know how many of these cases have yet to be identified.

TTP registries: Yoshihiro Fujimura (Japan)

• Central referral center at Nara University Medical Center. 882 thrombotic microangiopathy syndromes referred to date. 4% are due to USS. • ADAMTS13 assays (activity and antigen) and ADAMTS13 genetic analysis. • 37 Upshaw-Schulman Sydrome patients - clinical features described. Recurrent thrombocytopenia in childhood (78%) and 100% incidence of pregnancy-related thrombocytopenia. • Half-life of plasma ADAMTS13 - 2.4 days. Some USS patients develop severe reactions to plasma. Will need rADAMTS13. No ADAMTS13 antibodies seen. • One patient with ADAMTS13 level of 5% but no symptoms by adult life.

ADAMTS13-VWF interactions: Reinhard Schneppenheim (Germany)

• Variants of VWF with differential ADAMTS13 proteolysis. Can be either increased (type 2A and 2B VWD) or decreased. • Will often be identifiable by abnormal multimers. • Can also demonstrate this with the use of either the full-length or smaller VWF substrates (ie.A1-A3 domains). • Beginning to test some of the VWF substrates under shear influences. There should be a stringent analysis of the qualitative features of the VWF being evaluated.

Standardized Animals Models of VWD: Cecile Denis (France) and David Lillicrap (Canada)

• Type 3 VWD dogs and VWF KO mice. For in vivo evaluation of VWF variant function. • In VWD animal models FVIII:C levels are higher 20-50%. • Species specificity for GPIb binding and ADAMTS13 processing. • VWF reconstitution with murine VWF - hydrodynamic delivery. • With pLIVE vector (liver-specific) promoter – VWF expression for 4+ weeks. • Differences in multimer profiles in the animals expressing from hydrodynamically-delivered plasmid.

DDAVP Biological Responsiveness: Stefan Lethargen (Denmark)

• International study – study update – study is ongoing. Two stage study – biological response followed by clinical response. Two year follow up. 13 centers have enrolled 247 patients. • 182 type 1 VWD patients. 23 Type 2A VWD and 21 type 2M VWD. • 174 patients have completed biological response evaluation.

• Clinical evaluation in approx. 45 cases to date. Variable results. Bleeding cessation/DDAVP efficacy in 67/77 patients. 78 bleeding events in 25 patients. Mainly epistaxis and menorrhagia. Evaluation further complicated by use of antifibrinolytics.

Type 3 VWD ISTH-WFH Initiative: Augusto Federici (Italy)

• International study involving interaction with World Federation of Hemophilia. • Type 3 mutational background – heterogenous. No clear founder mutations. • Many unresolved issues for this rare disease. Goal to collect and analyze 500+ patients. Multiple clinical and laboratory parameters to be collated. • Even in type 3 VWD ~48% of patients may not bleed in one year. Unclear why there is clinical heterogeneity. • Initial funds obtained from Bayer Hemophilia Awards program to start this project. Will need additional funds to complete the project.

Prophylactic Treatment of VWD: Tom Abshire (USA)

• Organization of Prophylaxis Study Network. International multicenter study. • Multiple outcome analysis – clinical and quality of life assessments. Will look at a number of types of bleeding – menorrhagia, epistaxis, joint bleeds and GI bleeding. • Non-randomized dose escalation approach - starting dose 60 VWF:RCo units/kg then going up if bleeding continues. • Will include type 1 and type 2A VWD that are non-DDAVP responsive and type 3 VWD. • Strict bleeding type and frequency inclusion criteria. • Also some retrospective analysis from past 1-2 years. • Current status - 22 centers enrolled. 37 patients enrolled to date from 6 clinical centers.

VWF and VWD Registries and Nomenclature: Anne Goodeve (UK) and David Lillicrap (Canada)

• ISTH VWF Registry. Dan Hampsire is the University of Sheffield maintenance person. • Platelet-type VWD registry now on-line. 55 patients assessed. Type 2B VWD confirmed in 25-60% of cases. • Encouragement to keep submitting new information to the database. In particular the VWF SNPs need to be added. • Currently, 26% of mutations in the Database are type 1 VWD. 19% 2A, 6% 2B. • Encouragement to reduce number of “unclassified” variants. • Proposal for change in Reference sequence – use chromosome 12 reference sequence deposited in GenBank. • Proposal for SSC publication on the database. Hopefully to be published in the coming year. • Nomenclature issue – use of Human Genome Variation Sequence (HGVS) convention.

Submitted by D. Lillicrap

Women’s Health in Thrombosis and Haemostasis

4 July 2008 Vienna, Austria

Chair: Andra James, USA Co-Chairs: Margareta Blomback, Sweden; Benjamin Brenner, Israel; Jacqueline Conard, France; Sabine Eichinger, Austria; Barbara A. Konkle, USA; Claire McLintock, New Zealand; Claire S. Philipp, USA

Introduction and Overview – Andra H. James, MD, MPH

• Purpose and function of SCCs • What constitutes issues in women’s health in thrombosis and haemostasis – • Introduction of co-chairs

Educational Activities Overview of the 2009 ISTH meeting and proposed educational sessions

• Next year there will be general sessions that address women’s issues plus we will need to come up with three educational sessions (30 minutes each) for inclusion in our 4 hour meeting time (90 minutes of educational sessions, 150 minutes of meeting time). We don’t know yet what the general sessions will be, but when we find out, we will need to make sure that the topics we select to present during our SSC meeting will be complementary. • Topics that have already been suggested for educational sessions within our SSC meeting next year- Anticoagulants in pregnancy Reproductive tract bleeding in women with bleeding disorders • Other topics that were suggested at the July 4, 2008 meeting: Diagnosis of pulmonary embolism during pregnancy Hormone therapy: contraception (J. Conard) Hormone therapy: postmenopausal

Next International Symposium on Women’s Health Issues in Thrombosis and Haemostasis will be February 6-8, 2009 in Prague - Benjamin Brenner MD

Report on the Women’s Health in Thrombosis & Haemostasis Scientific Subcommittee (SSC) Inspired or Sponsored Publications – Claire Phillip, MD

• A screening tool for identifying women with menorrhagia for hemostatic evaluation – published this year in the Am J of Obstet Gynecol • A multi-site, prospective cross-over study of intranasal desmopressin and oral tranexamic acid in women with menorrhagia and abnormal laboratory hemostasis – presented on behalf of her co- investigators – manuscript in preparation

Update on Registries

• Pregnancy outcome in women with mechanical heart valves – presentation of large series of cases by Claire McClintock, MD – manuscript in preparation – proposal for data management by MedSciNet with funding from an unrestricted grant to ISTH • Mirena IUD for menorrhagia in women with bleeding disorders – registry is now on the ISTH website. Andra James reporting for Rezan Kadir, MD

Update on Ongoing international collaborations

• Menorrhagia in women affected by bleeding disorders – an international study – Flora Peyvandi MD presented an overview of the study. ISTH members are encouraged to participate. More information can be found at www.wrbd.org

New registry:

• Corpus luteum bleeding in women with bleeding disorders – a summary and a proposed registry was presented by Ron Hoffman MD. The data collection form is ready to go and we will get it posted to the ISTH website.

Presentation on haemostatic disturbances in women undergoing in vitro fertilization (IVF) – Eli Westerlund, MD

Topics suggested for inclusion in next year’s SSC meeting (besides the educational sessions):

• Update on Mirena registry - R. Kadir, MD • Update on mechanical valves registry – C. McLintock, MD • Data on fondaparinux in pregnancy – S. Duncan, MD • Update on the international study - Menorrhagia in women affected by bleeding disorders – F. Peyvandi MD • Update on registry of corpus luteum bleeding – R Hoffman, MD • Predictive scores for thromboprophylaxis in pregnancy – TBA • D-dimer levels in pregnancy – R Alkier, MD • Thrombin generation testing in pregnancy • Preliminary results of multicenter study of VWD levels in pregnancy – B Konkle, MD • Update in antithrombin registry – J Conard, MD • Proposed registry of women with thrombocythemia in pregnancy –

Submitted by A. James

Working Group on Coagulation Standards

4 July 2008 Vienna, Austria

Chair: A Hubbard (UK)

Review of Lot #3 (A Hubbard, NIBSC, UK)

An update on the stability assessment and issue of Lot #3 was presented. Stability of Lot #3 is being assessed through an accelerated degradation study which commenced in December 2003 and has involved testing at two time-points – after 2.2 years (February 2006) and after 4.2 years storage (February 2008). The residual potencies of four coagulation factors (FV, FVII, FVIII and FIX) have been measured in two laboratories (NIBSC and Royal Hallamshire Hospital, Sheffield, UK). Similar estimates were obtained in both laboratories and these have been combined for analysis of predicted degradation rates according to the Arrhenius equation. Mean predictions of loss for vials stored at -20 °C ranged from 0.002 % per year for factor VII to 0.069 % per year for factor V. The upper 95% confidence limits of predicted loss were consistent with a “shelf-life” of 28 years based on the stability of the most labile analyte, factor V. This indicates that Lot #3 is extremely stable when stored at -20 °C and also that the period of valid use for Lot #3 has probably been underestimated with the assigned expiry date of 2014.

A total of approximately 21,000 vials of Lot #3 have been issued over two years to 26 manufacturers and 2 quality assurance schemes. The remaining stock (approx 30,000 vials) will be used up by June 2011 based on an annual use of 9 – 10,000 vials. Considering the large calibration exercise required for 19 analytes it was suggested that we should consider obtaining Lot #4 by Q4 2009 in order to coincide with scheduled replacements of WHO International Standards for Factors II, VII, IX, X, plasma and Antithrombin, plasma. In order to reduce the frequency of calibration exercises it was suggested that a larger batch size should be considered for Lot #4 or alternatively, the simultaneous calibration of two different lots of plasma. The strategy for replacement will be discussed further with the SSC Executive.

Assignment of value for t-PA antigen (C Longstaff)

Lot #3 was included in the collaborative study for the WHO 1st IS t-PA antigen which was formally established in October 2007. It has now become possible to assign a value of 3.0 ng/ml to Lot #3. Dr Longstaff indicated that one of the kit methods used in the calibration may underestimate t-PA antigen through the inability to detect cryptic t-PA. Lot #3 with an assigned value for t-PA antigen was considered useful for the comparative evaluation/normalisation of test plasma samples even though the level of t-PA antigen in Lot #3 was too low to allow its use as a conventional reference through the generation of a calibration curve. There were no objections to the assignment of a value of 3.0 ng/ml for t-PA antigen. This proposal will be circulated to the Executive Board for approval.

Experience of EQA schemes with Lot #3

Use for trouble-shooting – UK NEQAS (S Kitchen)

A new use for Lot #3 involving the trouble-shooting of anomalous laboratory results associated with the UK NEQAS surveys has been undertaken for a trial period of one year. Samples of Lot #3 were dispatched to 12 laboratories primarily to investigate factor V and XI estimation (prior to assimilation of the new WHO International Standards for the calibration of commercial calibrants) and also for the assay of factor II, factor VIII, Protein C, Protein S and von Willebrand factor antigen. The use of Lot #3 resolved a discrepancy between two kit methods for Protein C activity, supporting the conclusion that the labeling of one kit reference was inaccurate. Another example demonstrated the resolution of a problem with VWF:antigen estimation linked to a specific lot of commercial reference which was subsequently withdrawn from use. There was general agreement that this use of Lot #3 should continue, providing the original conditions regarding areas of use and

67 vial quantities were followed. A report on the use by UK NEQAS will be submitted to the SSC executive with a request for continuation.

College of American Pathologists (M Cunningham)

Lot #3 was included in two surveys in 2008 relating to VWF estimation and thrombophilia testing. The results for most analytes were generally supportive of the assigned values for Lot #3 with most deviations less than 10% (FVIII:C, VWF:RCo, VWF:Ag, Antithrombin, Protein C and Protein S). The largest bias was seen with estimates from specific single kit methods for Protein C activity, Protein S activity and free Protein S antigen which differed from the assigned values by more than 20%.

Joint Committee on Traceability in Laboratory Medicine (E Gray)

Dr Gray described the aims and procedures of the JCTLM and how these related to Lot #3. The meeting was informed that Lot#3 had been nominated in Cycle IV and accepted on to the BIPM database of higher order reference materials in 2008.

Submitted by A. Hubbard

Working Group on Gene Nomenclature

A Standard System for Nomenclature of Genetic Variants in Thrombosis & Haemostasis;

Executive Summary

Anne Goodeve, Pieter Reitsma and John McVey, the International Society on Thrombosis and Haemostasis Scientific and Standardisation Committee Nomenclature Working Group

Summary A wide variety of different ways of referring to genes and sequence variations within them have been utilized. International groups have devised standard systems to bring order and standardisation to nomenclature, and these systems are being adopted by some investigators within the haemostasis community. Their piecemeal introduction will result in confusion. The ISTH SSC Nomenclature Working Group was convened in 2007 to examine sequence variation nomenclature and make recommendations to the ISTH. The group recommends that the standard systems for nomenclature are followed and suggests ways to facilitate their implementation.

Background Standardised systems for naming genes and variations within them have been devised and matured over the past 10 years. The Human Genome Organisation (HUGO) and International Federation of Human Genetics Societies (IFHGS) have overseen these developments and through their lead, the Human Genome Variation Society (HGVS) has devised an extensive scheme for nomenclature of sequence variants. Some fields have adopted these nomenclature schemes so that they have become commonplace. In other areas they have only been utilised to a limited extent, or not at all.

Many coagulation genes were cloned and sequenced during the 1980s and as a result, many genes and proteins have their own idiosyncrasies of naming and numbering, making it difficult for those unfamiliar with them to readily understand conventions used.

Drivers for change It is particularly important in molecular genetic diagnostic work that there is no confusion resulting arising from differences in mutation nomenclature between laboratories. Several external quality assessment (EQA) schemes require use of HGVS nomenclature for unambiguous patient reports, including the European Molecular Genetics Quality Network (EMQN) and UK National External Quality Assessment Scheme (UK NEQAS) for Molecular Genetics.

Many journals now require HGVS recommendations to be followed, although policing nomenclature use by reviewers is variable. von Willebrand factor (VWF) nomenclature was successfully altered in line with HGVS conventions following an ISTH-SSC on VWF recommendation in 2001.

Nomenclature system The HUGO Gene Nomenclature Committee (HGNC) curates gene names. Each unique gene symbol comprises a short representation of the descriptive gene name. Names contain only Latin letters and Arabic numbers and contain no punctuation or reference to species. Hierarchical series are used for both structural and functional gene families; e.g. F2, F5 and F8 and GP1BA, GP1BB and GP6.

Exon and intron numbering Numbering is sequentially from the 5’ end of the gene using Arabic numbers for both exons and introns. Letters are not used.

Gene and protein numbering Sequence variants differing from a specified reference sequence are recorded. Candidate mutations and polymorphisms are named using the same nomenclature. Prefixes denoting sequence type are used; cDNA uses c. and protein uses p.

DNA alterations The sequence is numbered from the A of the ATG initiator methionine as +1. Nucleotide alterations in cDNA follow the nucleotide number, to prevent confusion with amino acid alterations e.g. c.123A>T. Intronic numbering utilises the closest exonic nucleotide and numbers from that point, for example c.1234+2G>A.

Amino acid alterations The sequence is numbered from the first methionine of the protein as +1. Three letter amino acid codes are used in preference to single letter codes as they are less likely to be confused during transcription between documents; particularly important in clinical work. For publication purposes, single letter codes are acceptable. Amino acid designations are placed either side of the codon number, e.g. p.Gly123Ser or p.G123S. Superscript text indicating codon number and arrows are not used.

Trivial names and reference to older nomenclature Names such as the factor V Leiden mutation are unlikely to be entirely replaced, so it is recommended that both HGVS nomenclature and the trivial name are used together. e.g p.Arg534Gln (c.1601G>A), the factor V Leiden mutation (previously 1691G>A; Arg506Gln). To aid transition, both old and new numbering can be used together within a document, e.g. p.Ala416Ser previously referred to as Ala384Ser in antithrombin.

Reference sequences Any description of sequence variation should indicate the DNA and protein reference sequence (RefSeq) number and version used, along with the numbering convention. RefSeqs available via the NCBI Entrez Gene web page will be accessible via links on the ISTH website.

Mutation and polymorphism databases DNA and protein numbering on locus specific databases (LSDB) may require amendment, and some curators have agreed to add additional field(s) to enable both colloquial and HGVS numbering to be displayed.

Adoption of the proposal It is suggested that each ISTH subcommittee considers the proposed nomenclature alterations, with a view to quick adoption in principle.

Promotion of numbering changes A listing of information to be hosted on the ISTH website is being developed to provide information for each of about 50 coagulation and platelet genes and proteins on HGNC gene names, gene and protein properties, links to RefSeq and LSDB web pages etc, plus an indication of the alterations required in numbering with examples of commonly reported variants.

For manuscripts submitted to the Journal of Thrombosis and Haemostasis, editors will be requested to add a radio button to the manuscript comments section of the reviewer feedback page asking “Have recommendations been followed for gene names and sequence variations (HGNC and HGVS)?”

A manuscript describing altered nomenclature has been prepared for submission to JTH as an ISTH SSC communication, if the recommendation to change is accepted.

Submitted by Anne Goodeve