Identification, Pathogenesis, and Treatment of Factor Inhibitors

Welcome to another issue of Seminars in Thrombosis and Hemostasis, the final one for 2009. This issue focuses on coagulation factor inhibitors. Several considerations led to the preparation of this issue. Primarily, Seminars in Thrombosis and Hemostasis has not published an issue on coagulation factor inhibitors since 2002, and there have been several advances during the intervening period in our understanding of inhibitor development, as well as in treatment. In addition, external quality assurance (EQA) providers have conducted several cross-laboratory evaluations of laboratory performance in the identification of coagulation factor inhibitors over the past decade, and the findings of these studies are reported here. These articles therefore represent the state of the art in relation to the identification, pathogenesis, and treatment of coagulation factor inhibitors.

We begin this journey of discovery with two articles related to the development of factor inhibitors. In the first one, Gouw and van den Berg provide a cutting-edge review on the etiology of inhibitor development in hemophilia and the role of genetics and environment in this process. They begin by reminding us that the most important complication of the treatment of hemophilia patients is the development of inhibitory antibodies against the missing coagulation factor as provided for therapy. Because the most common form of hemophilia is hemophilia A, and the most common type of inhibitor is similarly that against factor VIII (FVIII), Gouw and van den Berg focus their review on this issue. Inhibitor development is caused by a complex interplay between both genetic and environmental factors. The risk of developing inhibitors is greatest in previously untreated patients with severe hemophilia A, and several genetic factors, such as a positive family history of inhibitors, ethnicity, and factor VIII genotype, and certain polymorphisms in immune modulatory genes are associated with an increased risk of inhibitor development. Several treatment-related aspects, including age of first treatment and intensive treatment with factor VIII for bleeds or surgery, have also been associated with a higher inhibitor risk. However, regular prophylaxis seems to have a protective effect on inhibitor development. The authors conclude that knowledge regarding the risk factors for inhibitor development enables better prediction and possibly even prevention in the development of inhibitors in patients with severe hemophilia A. However, much remains to be learned.

The second article continues the narrative related to the pathogenesis of FVIII inhibitors, and in particular Ananyeva and colleagues discuss recent advances in our understanding of the molecular mechanisms by which inhibitors inactivate FVIII and the experimental approaches used for the mapping of inhibitor epitopes. This has involved generating and evaluating the interaction between a large number of monoclonal antibodies against FVIII and a series of peptides reflective of various parts of the FVIII molecule. Ananyeva and colleagues also complement this discourse by providing an overview of genetic and environmental influences on inhibitor development, and to be more comprehensive they present a comparative analysis of treatment of hemophilia A patients with inhibitors with currently available bypassing agents: activated prothrombin complex concentrate (FEIBA VH, Baxter Healthcare Corp., Westlake Village, CA) and recombinant activated factor VII (rFVIIa). Finally, they describe some ongoing research programs aimed at developing new treatment options for these patients.

The next section of this issue deals with the laboratory detection of coagulation factor inhibitors. Accordingly, Verbruggen and colleagues first provide an up-to-date review on the laboratory detection of FVIII inhibitors, and they describe in detail the various problems encountered with the assays used in the quantification of functional FVIII inhibitors. All methods for the detection of these inhibitors are based on the measurement of inactivation of FVIII in a mixture of test plasma containing the putative inhibitor and an exogenous source of FVIII. Various types of assays have been developed since the first inhibitor was described in 1941.[1] This inhibitor was identified by the finding of a prolonged whole blood clotting time in combination with subsequent serious bleeding complications in a second inhibitor-free hemophilia patient following an infusion of blood from the inhibitor patient. These findings were confirmed in vitro by the observation of a prolonged clotting time in a mix of normal citrated whole blood and whole blood of the inhibitor patient compared with a mix of blood of an uncomplicated hemophilia patient and normal blood. Thankfully, we now have better and safer systems in place for the identification of factor inhibitors, including the two methods most often used, namely the Bethesda assay and the Nijmegen assay.[2] [3] Although the Nijmegen assay shows better specificity and lower intra- and interlaboratory variation, it is still hampered by several limitations related to assay characteristics. These include pH, assay temperature, time of incubation, formulation of the control sample, and the von Willebrand factor (vWF) content of the assay mixture. Epitope specificity also plays an important role in the reliability of functional assays because inhibitors against the C2 domain of FVIII are more difficult to quantify compared with inhibitors against the A2 domain. Finally, lupus anticoagulants (LAs) can interfere with inhibitor assays, resulting in aberrations.

The next article from Kershaw and colleagues discusses the laboratory identification of factor inhibitors from the perspective of a large tertiary hemophilia center, perhaps the largest such center in Australasia. The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titers during the course of inhibitor eradication therapy. This laboratory performs >200 inhibitor evaluations per year. Their overview examines the behavior of various coagulation inhibitors and laboratory tests with an emphasis on the Bethesda assay for factor inhibitors, and the authors provide a very practical synopsis of the laboratory test process, including potential pitfalls. They advise that the identification of coagulation factor inhibitors by the hemostasis laboratory requires a careful and systematic approach that initially excludes other possible causes of prolonged screening tests such as the activated partial thromboplastin time (aPTT) and prothrombin time (PT). Once the laboratory is confident that a specific coagulation factor inhibitor is present in a test sample, its strength or titer must be measured because the treating clinician will use this information as a guide for therapy. This article discusses inhibitors that develop in hemophilia as a consequence of an immune response to factor replacement therapy, as well as those arising as an autoantibody leading to the condition of acquired hemophilia. Although the most common presentation of inhibitors are those directed against FVIII, this article also discusses other factor inhibitors such as those directed to factor IX (FIX). The Bethesda assay, the most widely used test for measuring the FVIII inhibitor titer, has several components that must be carefully controlled to achieve consistent results. The article also discusses several other issues related to the appropriate identification of factor inhibitors.

Tay and coworkers, from a second hemophilia center within Australia, then provide a personal glimpse of their recent experience of acquired hemophilia A, a rare and serious bleeding disorder where prompt and correct diagnosis is crucial and immune suppression is often required for FVIII autoantibody eradication. Their title includes the poignant words “trials and tribulations” and highlights some of the highs and lows related to the identification and management of these individuals. Expensive bypassing agents such as rFVIIa may be required to treat clinically significant bleeding. They retrospectively identified 25 patients during a 12-year period (1997 to 2008) and reviewed diagnostic features, treatment for bleeds and used to eradicate the autoantibody, treatment response, and survival outcomes. The incidence in their geographic region was 1.20 cases per million per year with a median age of 78 years with an approximately equivalent sex ratio (12 males versus 13 females). Overall mortality was 25% (n = 6), and advanced age and lack of treatment were predictive of poor survival outcomes. They conclude by saying that the very elderly (>75 years of age) may warrant a different treatment modality to improve outcomes in this group.

The next series of articles investigates and compares the laboratory testing of factor inhibitors between laboratories, as identified through various EQA studies as undertaken by several EQA providers. The first article is by the United Kingdom NEQAS (National External Quality Assessment Scheme) for Blood Coagulation, the EQA provider active in this process for the longest period of time. They report the results of EQA exercises in which 60 to 120 centers performed FVIII inhibitor testing on a series of samples over a 13-year period. Samples from seven different subjects were distributed for analysis including four different subjects with severe hemophilia A with antibodies following replacement therapy, one subject with acquired hemophilia A and antibodies to FVIII, one subject with normal FVIII and an easily detected LA, and one subject with mild hemophilia A and a difficult-to-detect LA but without antibodies to FVIII. In all of the surveys, the results obtained in different centers analyzing the same sample varied to the extent that would have influenced patient management decisions, and there was considerable interlaboratory variation in the results of FVIII inhibitor testing that did not improve over the survey period. The coefficient of variation (CV) of results in different centers was between 33% and 106% using samples from patients with severe congenital hemophilia A and inhibitors. In some cases, results were affected by assay components. For example, for one test plasma the mean FVIII inhibitor results in centers using one source of normal plasma was 3.9 Bethesda units [BU]/mL compared with a mean of 5.7 BU/mL in centers using a different normal plasma source (p = 0.04). In summary, the NEQAS group concludes that the detection of FVIII inhibitors is not the same in different centers, the degree of variability noted makes it likely that assay variability has contributed to the lack of international consensus in relation to the real incidence of FVIII inhibitors in different clinical settings, and improvements in assay standardization are urgently needed.

The second EQA-related article comes from the European Concerted Action on Thrombosis (ECAT) Foundation. This study includes the results of six different surveys for the period 2006 to 2008 with 100 to 170 participating laboratories, and so it reflects the findings of another large EQA program, albeit over a shorter period of time. The overall between-laboratory variation ranged from 28% to 52% with a slightly lower variation for the Nijmegen assay (~39% on average) than for the Bethesda assay (~45%). This report also assesses the effect of differential laboratory practice on interlaboratory variability. Thus the use of buffered normal pooled plasma as an FVIII source showed better performance compared with the use of nonbuffered pooled plasma; likewise the use of FVIII-deficient plasma compared with the use of imidazole buffer. However, the combination of both appeared essential for lowest between-laboratory variation. The Nijmegen assay also showed better performance with respect to specificity and sensitivity than the Bethesda assay, although the results for neither were entirely satisfactory. In general, Meijer and Verbruggen conclude that the measurement of FVIII inhibitory antibodies with the Nijmegen assay should be favored over the use of the Bethesda assay, but that further improvement of the laboratory test for FVIII inhibitors is also urgently needed.

The final article related to EQA in this issue comes from the Royal College of Pathologists of Australasia (RCPA) Haematology Quality Assurance Program (QAP). This article reflects in part on the experience of this EQA in regard to laboratory identification of coagulation factor inhibitors, and in part it reviews the composite of data available from all the EQA studies, including those previously published[4] [5] [6] as well as those within the current issue of Seminars in Thrombosis and Hemostasis. They discuss in particular the cross-EQA laboratory data on the most commonly seen factor inhibitors (i.e., those directed against FVIII), as differentially detected either using the original or Nijmegen-modified Bethesda assay. In addition, Favaloro and colleagues identify several circumstances that can arise in which the laboratory may be faced with test samples that potentially reflect false identification of factor inhibitors. These include LA and other events generally related to preanalytical variables, including incorrect sample presentations.[7] This article reviews each of these elements, again largely from the perspective of EQA studies. Their analysis reveals a wide variety of test practice among inhibitor-testing laboratories, a wide variation in detected inhibitor levels in cross-tested samples, and substantial evidence of false-positive and false-negative detection of factor inhibitors. Each of these findings holds significance for the clinical management of patients affected by these inhibitors. In agreement with the ECAT Foundation, the RCPA QAP also reports that Nijmegen methods appeared to do better than Bethesda methods, with lowered interlaboratory CV and less likely false-negative findings. They conclude that there is still much need for standardization and improvement in factor inhibitor detection, and with the hope that such reports provide a basis for future improvements in this area.

The final three articles discuss the management of people with inhibitors. The first of these, by Franchini and Lippi, broadly discusses the most recent options available for treating patients with acquired hemophilia and congenital hemophilia with inhibitors. The mainstay of therapy in patients with congenital hemophilia is factor replacement. However, the development of inhibitors in these patients is a major complication that represents an important challenge in hemophilia care. Development of inhibitors complicates the clinical course of severe hemophilia in up to 30% of patients with hemophilia A and up to 5% of patients with hemophilia B. Although the main short-term objective of the treatment of alloantibodies against FVIII and FIX is to control the bleeding diathesis, the eradication of the inhibitor is the leading long-term goal. The management of severe bleeding episodes and the definitive eradication of the autoantibody are also the two main options for the clinical management of patients with acquired hemophilia, a rare but life-threatening hemorrhagic condition. All of this is addressed in this state-of-the-art review.

The second in this series of management articles by Franchini and coworkers focuses more specifically on the role of rFVIIa in the prophylaxis of congenital hemophilia in people with inhibitors. In brief, the development of inhibitors against therapeutically administered FVIII or FIX is the most challenging complication of hemophilia patients with inhibitors, and the introduction of bypassing agents (i.e., activated prothrombin complex concentrates and rFVIIa) has dramatically improved the management of bleeding episodes in such patients. Over the last decade, there have been increasing reports on the ability of bypassing agents to prevent surgical, joint, or other bleeds in inhibitor patients. Among these bypassing agents, rFVIIa is currently receiving the most attention.

The final article both in this series and in this issue of Seminars in Thrombosis and Hemostasis is on the management of hemophilia in patients with inhibitors from the perspective of developing countries. Mathews and coworkers review the limited data on inhibitors in patients with hemophilia in developing countries. There is a perception that the overall prevalence of inhibitors, ranging from 7% to 19% in different reports, may be lower in these countries as compared with that reported from developed countries. This may be possible given the fact that most patients are treated after 2 years of age with plasma-derived clotting factor concentrates. Whether genetic or other environmental factors also contribute to this requires further evaluation. According to these workers, there is also a need to develop laboratory infrastructure and establish quality control programs for laboratory tests for inhibitors in developing countries. Management options vary widely given the socioeconomic diversity among these countries. Significant individualization of approach to management is therefore required depending on the available resources, particularly with regard to the use of bypassing agents, a situation that differs considerably from that of developed countries. The limited data on immune tolerance induction with some low-dose regimens in developing countries also deserve further evaluation. Nevertheless, these workers conclude that even in resource-constrained environments, education and a policy of systematic screening of patients associated with judicious use of bypassing agents can significantly improve the care of patients with hemophilia who develop inhibitors.

I hope that you enjoy reading the collection of articles in this issue of Seminars in Thrombosis and Hemostasis, and I would like to express my gratitude to the respective authors for their interesting and timely contributions.

REFERENCES

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• 3 Verbruggen B, Novakova I, Wessels H, Boezeman J, van den Berg M, Mauser-Bunschoten E. The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability.  Thromb Haemost. 1995;  73(2) 247-251
• 4 Favaloro E J, Bonar R, Duncan E et al.. Identification of factor inhibitors by diagnostic haemostasis laboratories: a large multi-centre evaluation.  Thromb Haemost. 2006;  96(1) 73-78
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