Blood coagulation is a rapid series of complex reactions, which triggers ‘haemostasis’ – a process by which the body responds to prevent blood loss. This occurs by a blood clot forming in a blood vessel at the site of an injury.
A series of proteolytic reactions takes place with a stepwise activation of coagulation factors – the ’coagulation cascade’ – which ultimately results in a fibrin clot forming.
Coagulation disorders can lead to excessive bleeding – this happens with haemophilia A – or excessive clotting such as in deep vein thrombosis.
Our laboratory focuses on the diagnosis and treatment of bleeding coagulation disorders, in particular haemophilia A and diseases relating to clot formation.
We carry out the Institute’s core functions of standardisation and therapeutic product control testing in these areas. Both of these functions are underpinned by related research and developmental activities.
The complex structure and function of coagulation factors – coupled with their low concentrations in blood plasma – makes it virtually impossible to quantify them by physico-chemical means.
The estimation of coagulation factors therefore relies on the principle of comparative bioassay relative to a reference standard containing a known amount of analyte. Reference standards prepared locally can provide consistency and continuity of testing within a single laboratory but do not address the issue of harmonisation in testing between multiple laboratories.
International Standards (IS), prepared by NIBSC and established by the World Health Organization (WHO) fulfil this role by providing a common single route of calibration for all local and secondary working reference standards.
The first IS for a coagulation factor – factor VIII (FVIII) – was established in 1971 in response to the need for harmonisation in the potency labelling of the ‘new’ therapeutic concentrates. This approach has subsequently been applied to most plasma coagulation factors.
Our laboratory’s primary responsibility is for standardisation of FVIII – both plasma-derived and recombinant – in therapeutic concentrates, in particular for the development of new and upkeep of current WHO IS and British Standards for FVIII Concentrate.
The current WHO 8th IS (07/350) for FVIII concentrate is the only one in a series of ISs for which there is a complete agreement in FVIII potency between one-stage clotting and chromogenic methods.
However, as new modified/longer-acting FVIII products emerge, there have been assay discrepancies and standardisation issues which will need to be addressed.
We have also carried out a series of field studies on behalf of the FVIII/FIX subcommittee of the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardisation Committee (SSC) to assess any standardisation issues relating to assays on licenced FVIII products.
These studies perhaps give a truer picture about how assays perform routinely in laboratories where local methodologies, standards and calculation of results are used to estimate potencies in FVIII concentrates.
As a European Official Medicines Control Laboratory (OMCL), Official Control Authority Batch Release (OCABR) testing of FVIII therapeutic concentrates is carried out as part of the Institute’s statutory function.
These products consist of freeze-dried, plasma-derived FVIII purified from large pools of plasma from donors. The plasma-derived FVIII concentrates will have undergone viral inactivation and are used primarily for replacement therapy in treating haemophilia A patients.
They are also used in immune tolerance induction (ITI) regimens to treat patients that develop antibodies or ‘inhibitors’ – alloantibodies or autoantibodies – to the FVIII molecule.
Batch release testing of FVIII products has been increasing at NIBSC over the past 10 years and is carried out for marketing in both EU and non-EU countries.
Haemophilia A is a recessive X-linked bleeding disorder caused by a deficiency or a functional abnormality of coagulation co-factor FVIII.
It affects 1 in 5000 males resulting in bleeding in joints, muscles and soft tissues. It is usually treated by prophylactic infusions of FVIII therapeutic products.
However, a major complication of such substitutive treatment is the development of an immune response – developing neutralising antibodies – towards the FVIII molecule, with most of the alloantibodies developed being directed to the A1, A3 and C2 domains of the FVIII molecule. Antibodies inhibiting FVIII activity – ‘inhibitors’ – preclude further use of FVIII, leaving patients in a life-threatening situation. About 30% of severe haemophiliacs have these antibodies.
FVIII autoantibodies can also develop in non-haemophiliacs, in a variety of clinical settings such as the postpartum period and chronic lymphocytic leukaemia.
Using recombinant FVIII has not reduced the incidence of inhibitors, suggesting that the forthcoming gene therapy for haemophilia may not reduce it either.
Furthermore, the methods used to prepare FVIII or reduce viral transmission carry the risk of altering the immunogenicity of the molecule.
Present treatments to eradicate FVIII inhibitors are unsatisfactory. They rely on methods such as infusion of large doses of FVIII – alone or in combination with non-specific immunosuppressive agents.
The cost of these treatments is prohibitively high and they tend to be effective only in patients with recent, low-titre, inhibitors.
Inhibitor development in up to 20% of patients is related to the gene defect – intron 22 inversion of the FVIII gene. Preventing inhibitors from developing is currently not feasible, as criteria to identify patients at risk are not yet fully defined.
There is therefore, a need for alternative, more efficient therapies which would be both specific and cost-effective. But before this can happen, we need to understand precisely the mechanisms of inhibitor development and by which FVIII interacts with other analytes in blood.
In addition, current methods to measure FVIII inhibitors are unsatisfactory and developing new assays which can measure inhibitors accurately and reproducibly is critical for any meaningful data for diagnosis and therapy.
We are working on a number of different research areas to address these problems:
Dr Sanj Raut - Principal ScientistAndrew Riches
07/350: 8th IS Factor VIII, concentrate09/264: 3rd IS Fibrinogen, Plasma09/242: 2nd IS Fibrinogen concentrate02/206: 1st IS Factor XIII, Plasma06/172: 1st NIBSC Working Standard for FEIBA10/188: 13th British Standard Factor VIII, conentrate