Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review

Inhibitors are antibodies directed against haemophilia treatment products which interfere with their function. Factor VIII (FVIII) inhibitors in haemophilia A and factor IX (FIX) inhibitors in haemophilia B are significant clinically when they require a change in a patient's treatment regimen. Their persistence may increase morbidity and mortality. Multiple laboratory tests are now available for detecting and understanding inhibitors in haemophilia. Inhibitors are traditionally measured by their interference in clotting or chromogenic factor assays. They may also be detected using immunologic assays, such as enzyme‐linked immunosorbent assay or fluorescence immunoassay. Anti‐FVIII or anti‐FIX antibodies of IgG₄ subclass best correlate with the presence of functional inhibitors. Improvements in inhibitor measurement have been recently introduced. Preanalytical heat treatment of patient specimens allows testing of patients without delaying treatment. Use of chromogenic and immunologic assays may aid in identification of false‐positive results, which are frequent among low‐titre inhibitors. Validated reagent substitutions can be used to reduce assay cost. New methods for defining assay positivity and reporting low‐titre inhibitors have been suggested. Challenges remain in the areas of quality control, assay standardization, monitoring of patients undergoing immune tolerance induction therapy and testing in the presence of modified and novel treatment products.     

Pharmacokinetics of factor VIII and factor IX

Summary.  A survey of principal pharmacokinetic (PK) studies on factor VIII (FVIII) and factor IX (FIX) plasma- and rDNA-derived concentrates, analysed by means of the PKRD program, has been performed. Notwithstanding the accurate definition of the study design, released in 1991 by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC-ISTH), a large variability of PK parameters has been pointed out. In the majority of the PK studies, the size of the population is small. In this situation, a careful individualization of haemophilia therapy is strongly recommended. The tailored prediction of loading and maintenance dosages and the need for strict control of trough FVIII/IX levels are mandatory not only to decrease the risk of bleeds but also to spare financial resources. Recently, the old problem of FVIII assay standardization has again become a concern among physicians, especially after the introduction of B-domain deleted rFVIII concentrate. The discrepancies between the widely used one-stage clotting assay and the chromogenic substrate assay seem to be solved by the introduction of a product-specific laboratory standard.     

The prevalence of factor VIII inhibitors and genetic aspects of inhibitor development in Chinese patients with haemophilia A

Summary. The prevalence of inhibitors in Chinese haemophiliacs has not yet been reported. The aim of this study was to identify the prevalence of factor VIII (FVIII) inhibitors among haemophiliacs who are treated only with plasma‐derived FVIII (pdFVIII), cryoprecipitate or fresh frozen plasma (FFP), and tried to explore the relationship between the generation of inhibitors and particular FVIII deficiency genotypes. Clinical information and blood samples of 1435 patients with haemophilia A (HA) were collected by six haemophilia centres in China. The Nijmegen modification of the Bethesda assay was used to detect inhibitors. Multiplex PCR, long‐range PCR and direct sequencing were performed for genotyping. The overall prevalence of inhibitors in Chinese HA patients was 3.9% and the prevalence of severe haemophiliacs was 4.3%; 18 of the 56 patients with inhibitors had high titres. A total of 38 different mutations were identified in the 55 patients with inhibitors, including 15 intron 22 and 3 intron 1 inversions, seven large deletions, 14 small deletion/insertions, seven nonsense mutations, one splice site mutations and eight missense mutations. Of 38 mutations, 28 were novel. Patients with large deletions and nonsense mutations were prone to have high titre inhibitors, with incidence rates of 57.1% (4/7) and 42.9% (3/7), respectively. In conclusion, the prevalence of inhibitors in Chinese HA patients is much lower than that reported for other ethnic groups and the large deletion and nonsense mutations are high risk factors for high titre inhibitor development.     

Porcine factor VIII provides clinical benefit to patients with high levels of inhibitors to human and porcine factor VIII

The development of an inhibitor against Factor VIII is an important complication of haemophilia and occurs in approximately 31% of patients [1]. Despite various approaches to their management, the presence of these inhibitors remains a major cause of morbidity and mortality. Inhibitors may be low level [<5 Bethesda Units (BU)] or high level [>10 BU]. Low-level inhibitors usually remain low and do not respond to administered factor VIII with a rise of the inhibitor titre; high-level inhibitors, in contrast, are characterized by a rapid rise in titre following treatment with factor VIII. Modalities of treatment of acute bleeding episodes in patients with inhibitors include 'overcoming' the inhibitor with very large doses of factor VIII, 'bypassing' the inhibitor blockade with products such as activated prothrombin complex concentrates or recombinant factor VIIa, 'removing' the inhibitor by plasmapheresis or immunoabsorption and 'repressing' it with immunosuppressive drugs and immune tolerance induction. An alternative approach is to use a porcine factor VIII concentrate which does not cross-react with the human factor VIII inhibitor. Following treatment with porcine factor VIII, functional factor VIII can be detected and the therapeutic levels correlate with cessation of bleeding. The use of porcine factor VIII may, however, result in the development of antiporcine factor VIII antibodies, limiting its use. Experience in patients with high-titre inhibitors indicates that porcine factor VIII therapy could be used in the presence of factor VIII inhibitors [2-11], including inhibitors to porcine factor VIII [5,7], and that haemostasis may be obtained in the absence of detectable levels of circulating factor VIII [7,11].     

Prophylactic dosing of factor VIII and factor IX from a clinical pharmacokinetic perspective

Summary.  The high cost and limited availability of factor concentrates make dosing of factor VIII (FVIII) or factor IX (FIX) a crucial issue in the prophylactic treatment of haemophilia. It has often been recommended that this treatment should aim to maintain a minimum plasma level of 1% of normal coagulation factor activity (FVIII:C or FIX:C). The dosage needed is commonly given as 25–40 U kg⁻¹ three times weekly for FVIII or twice weekly for FIX. However, these guidelines are valid only with several qualifications. First, the actual trough levels required may vary considerably between patients. The clinical severity of haemophilia may depend on more factors than the endogenous level of FVIII:C or FIX:C. Secondly, interindividual variations in dose requirements are also due to variance in the pharmacokinetics of the coagulation factors. Pharmacokinetic calculations are useful to design optimal dosing schedules to achieve required trough levels of FVIII:C or FIX:C. Moreover, tailoring of the dosing of FVIII or FIX according to their disposition in the individual patient can markedly improve the cost-effectiveness of prophylactic treatment. However, the usefulness of in vivo recovery as a guide for prophylactic dosing seems questionable. It should be clearly understood that maintaining a certain trough level of FVIII:C or FIX:C is not an end in itself. Clinical outcome, not the achieved trough level, determines whether a dosage is adequate. Chiefly for economic reasons, the minimum effective dosage of coagulation factor should be determined and used in every patient. The dose requirement should also be re-evaluated at appropriate times.     

Combined prednisolone and intravenous immunoglobulin treatment for acquired factor VIII inhibitors: a 2-year review

Acquired inhibitors to factor VIII (FVIII) are rare, but life-threatening in up to 22% of cases. The optimal therapy for suppression of these inhibitors remains unclear. Prednisolone is the mainstay of therapy, producing responses in approximately 30% of cases. Intravenous immunoglobulin (IVIg) has a similar response rate, but a more rapid effect. We report the results of prednisolone 1 mg kg(-1) combined with IVIg 2 g kg(-1) in divided doses as first-line therapy in seven consecutive patients with acquired FVIII inhibitors. All patients were bleeding at the time of diagnosis with prolonged activated partial thromboplastin time. There were four complete responses, one partial response, one nonresponse and one with an inadequate follow-up for assessment of response, giving an overall response rate of 71%. In all complete responders the inhibitor declined rapidly and was undetectable by day 21 from start of treatment. Therapy was well tolerated and responses have been maintained off treatment for 2-8 months. This is a safe, well-tolerated rapidly acting regimen with good response rates.     

Immunosuppressive Treatment in Haemophiliacs with Inhibitors to Factor VIII and Factor IX

9 patients with severe haemophilia A and inhibitors (inhibitor levels between 0.1 to 5.8 U/ml) and 3 patients with severe haemophilia B and inhibitors (inhibitor levels between 0.1 to 11 U/ml) were treated on a total of 16 and 13 occasions, respectively, with a large dose of antigen (factor VIII or factor IX) and cyclophosphamide (10–15 mg/kg b.w. i.v. initially and then 2–3 mg/kg b.w. orally for 7–10 days) in connection with severe bleeding and surgery. All the patients had proved not to respond to treatment with factor VIII or factor IX concentrate alone, and all except one had shown strong secondary antibody increases. In 6 of the patients with haemophilia A the treatment (11 occasions) had a satisfactory haemostatic effect and even permitted neurosurgery without bleeding complications. The inhibitor level remained at zero for 5–10 days, after which it gradually began to return towards its original level. In these cases it was possible to give factor VIII in amounts which neutralised the inhibitor and afterwards raised the factor VIII initially to at least 50%. In the 3 patients with haemophilia B treatment (13 occasions) was successful except on one occasion, and surgery was performed without abnormal bleeding. The factor IX level was initially raised to at least 50% except in the one failure. The inhibitor level remained at zero for 12 days to 3 months, after which it gradually rose towards its original level. One patient was treated on 8 occasions.     

Resistance to Activated F IX Concentrate (FEIBA)

A 10-year-old haemophiliac with an inhibitor to Factor VIII was treated by repeated infusions of an activated Factor IX concentrate (FEIBA). During therapy the patient was monitored with extensive coagulation studies, and the initial effect of FEIBA on the whole blood coagulation time was seen to disappear in spite of increased doses of concentrate. A fall in Factor IX activity and a rise of antithrombin III and anti-Factor Xa was recorded, while the titer of anti-F VIII remained unchanged. The exact nature of this FEIBA-resistant state was not revealed, however, it was speculated that a rise of natural inhibitors of the coagulation system could explain the findings.     

Immune tolerance in children with factors VIII and IX inhibitors: a single centre experience

Alloimmune FVIII and FIX inhibitors are the most serious complication of haemophilia in the postviral contamination era and their optimal management remains controversial. We present 15 boys with severe haemophilia (14 with haemophilia A and 1 with haemophilia B) who have received immune tolerance at our centre over a 9-year period. Twelve of them (80%) were successfully tolerized with varying dose intensities, but three of them (including the boy with haemophilia B) failed tolerization. The factors, which were associated with successful tolerance in our group, were a low maximum inhibitor titre and a short interval between diagnosis of the inhibitor and the start of immune tolerance. The time taken to achieve immune tolerance varied from 1 to 27 months and none of the inhibitors have recurred. Two of the three boys who failed immune tolerance had had their inhibitor for 72 and 69 months, respectively before tolerance was attempted.     

Role of enhanced half‐life factor VIII and IX in the treatment of haemophilia

Treatment of congenital haemophilia with factor VIII and IX concentrates often requires frequent infusions. This has obvious implications in establishing effective administration strategies and, in turn, adherence. To overcome these issues, three main technologies – polyethylene‐glycol, Fc‐neonatal IgG₁ and albumin fusion products – have emerged into various stages of clinical development. Published data indicates an approximately 1·5‐ and fivefold increase in half‐life of factor VIII and IX, respectively, compared to standard recombinant concentrates. Studies into efficacy and safety are starting to be published. Monitoring and optimal use of these new concentrates remains unknown. Weekly factor IX prophylaxis appears to be a feasible prophylactic regimen in haemophilia B patients. Weekly longer‐acting FVIII is unlikely to provide adequate prophylaxis in most patients with haemophilia A but may reduce the frequency of infusions. Ongoing clinical trials and real life experience will help shape how these products can be used in practice and their cost effectiveness. The drive for convenience however should not overshadow the ultimate goal of prophylaxis, namely, preventing bleeding and arthropathy.     

Cross-reactivity to porcine factor VIII of factor VIII inhibitors in patients with haemophilia in Australia and New Zealand

Abstract Background: Inhibitory antibodies which neutralise factor VIII develop in 10–20% of individuals with inherited haemophilia A and rarely as autoantibodies in normal individuals to cause acquired haemophilia. The antibodies are directed against human factor VIII but cross-react to varying degrees with porcine factor VIII. Porcine factor VIII can be used for treatment in individuals with low cross-reactivity.
Aims: To determine the cross-reactivity of factor VIII inhibitors between human factor VIII and porcine factor VIII, in a population of patients with inherited and acquired haemophilia A. Also, to determine whether patients with inherited haemophilia and inhibitors have a higher incidence of factor VIII gene inversion in intron 22.
Methods: Samples and data sheets from 43 patients with inherited and ten with acquired haemophilia were submitted from hospitals in Australia and New Zealand. Inhibitor levels to human and porcine factor VIII were measured by the Bethesda method in 39 with inherited and nine with acquired haemophilia A.
Results: Of 39 patients with inherited haemophilia A, cross-reactivity was 0% in 17 patients,1–19% in six, 20–39% in 11 and 40–80% in five. In six of nine patients with acquired haemophilia cross-reactivity was 7%. In inherited severe haemophilia A, the frequency of the intron 22 inversion was not greater in 37 study patients than in 28 patients without an inhibitor.
Conclusions: Many patients in Australia and New Zealand with inhibitors to human factor VIII presently show a low or absent level of cross-reactivity to porcine factor VIII. These may respond to treatment with this concentrate at least in the short term. There remains a group of patients with high cross-reactivity who will respond only to recombinant factor Vila or prothrombin complex concentrates.     

Breastfeeding does not influence the development of inhibitors in haemophilia

Our aim was to test the hypothesis that breastfeeding may reduce development of inhibitors in male infants with haemophilia by inducing an oral immune tolerance to factor VIII. To achieve that goal, we performed a structured epidemiological survey comprising all males born with severe haemophilia A (in all 100 patients, 19 with inhibitors) or haemophilia B (in all 16 patients, six with inhibitors) in Sweden in 1980-99. Our results show no protective effect of breastfeeding.     

Low-dose activated factor IX complex concentrates (FEIBAR) for post-operative haemostasis in a patient with high responding factor VIII inhibitors

Replacement therapy in patients with severe haemophilia A is associated with the development of inhibitory antibodies in about 15% [1,2]. The presence of inhibitors of factor VIII greatly complicates and compromises the treatment of these patients because of the lack of any completely satisfactory product to treat them. Haemostatic management for surgery in patients with inhibitors is very difficult. A product frequently used to treat bleeding episodes in such patients is prothrombin complex concentrate (PCC) [3] or its activated derivative (APCC) [4]. Activated recombinant human factor VII is another option [5]. For both these modalities of treatment, there are no laboratory tests that can be used to monitor clinical efficacy [5, 6]. Porcine factor VII is therefore the preferred product for surgery in patients with high-responding factor VIII inhibitors [7]. Unfortunately, none of these products are readily available in most developing countries, including India.
We report the management of a patient with high-responding factor VIII inhibitor using low doses of FEIBA^R (Immuno, Austria) in the post-operative period.     

Immune regulatory gene polymorphisms as predisposing risk factors for the development of factor VIII inhibitors in Indian severe haemophilia A patients

Development of inhibitors to factor VIII, a serious complication of replacement therapy in haemophilia A patients, leads to increased bleeding, morbidity and mortality. There is no data on the risk factors for inhibitor development in Indian patients with severe haemophilia A. Our aim was to study the role of immune regulatory gene polymorphisms in the development of inhibitors. Fourteen immune regulatory gene polymorphisms (IL1β, IL4, IL10, TNFA and CTLA4) were analysed in 120 patients with severe haemophilia A, i.e. 50 inhibitor positive patients, and 70 inhibitor negative control patients, by PCR-RFLP, DNA sequencing and allele-specific PCRs. The IL10 promoter 'GCC' haplotypes overall (P: 0.002, OR: 3.452, 95% CI: 1.607-7.416), and 'GCC/ATA' (P: 0.011, OR: 3.492, 95% CI: 1.402-8.696) haplotype, associated with high and intermediate IL10 production, respectively, were significantly higher in inhibitor positive patients, whereas the 'non-GCC' haplotypes overall (P: 0.002,OR: 0.290, 95% CI 0.135-0.622) and 'ATA/ATA' haplotype (P: 0.025, OR: 0.278, 95% CI: 0.096-0.802), associated with low IL10 synthesis, were significantly higher among inhibitor negative patients. The TNFA rs1799724 C/T heterozygote prevalence was significantly higher in the inhibitor positive group (P: 0.021, OR: 3.190, 95% CI: 1.273-7.990), whereas the other polymorphisms showed no statistically significant association with the presence of inhibitors. Different immune regulatory gene polymorphisms play a significant role as possible risk factors for the development of inhibitors in severe haemophilia A patients.     

A Bayesian approach to the assessment of inhibitor risk in studies of factor VIII concentrates

The assessment of inhibitor risk is a crucial component in the clinical development of new and modified factor VIII (FVIII) preparations. There has been a recent discussion about the design of studies and the assessment of inhibitors and inhibitor risk in such studies at a recent FDA-sponsored FVIII Inhibitor Workshop, and new requirements for the success of these trials have been proposed to evaluate inhibitor data based on the use of an upper 95% confidence bound. We review the consequences of these requirements and demonstrate that for any product to succeed, it must have an extremely low underlying risk of inhibitor development. Furthermore, several existing commercially available FVIII products with an excellent safety record would not necessarily pass these endpoints. As a result, we propose an alternative set of acceptance criteria based on a Bayesian statistical paradigm. This approach is based on the determination of a probability that the product in question actually has an inhibitor risk below some pre-set limit, a concept that we believe is more intuitive than the traditional confidence interval method. We show that all existing products would pass this approach, but a product (Bisinact) with known inhibitor risk would not.     

The use of an activated factor IX complex (Autoplex) in the management of haemarthroses in haemophiliacs with antibodies to factor VIII

A new activated factor IX product (Autoplex) has been used for the treatment of 18 haemarthroses in three haemophiliac patients with antibodies to factor VIII and a history of high anamnestic response to challenge with factor VIII. Results were evaluated by assessing pain, tenderness, range of movement and girth at 8 and 24 hours. A score of 2 was given for improvement at 8 hours and 1 for improvement at 24 hours. The result was expressed as a percentage of the possible score. Nine episodes treated with less than 35 units of Autoplex/kg had a mean score of 52% and nine episodes treated with more than 35 units/kg had a mean score of 73%. When only the more severe group of bleeds was considered, an even more marked dose related response emerged. When the ratio of the percentage fall in the PT to the percentage fall in the KCCT was plotted against the outcome, a significant correlation emerged (r=0.43; P= 0.02).