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Distinguishing lupus anticoagulants from factor VIII inhibitors in haemophilic and non-haemophilic patients.

ABSTRACT: INTRODUCTION:Accurate diagnosis of an inhibitor, a neutralizing antibody to infused factor VIII (FVIII), is essential for appropriate management of haemophilia A (HA). Low-titre inhibitors may be difficult to diagnose due to high rates of false-positive inhibitor results in that range. Transient low-titre inhibitors and false-positive inhibitors may be due to the presence of a lupus anticoagulant (LA) or other non-specific antibodies. Fluorescence immunoassay (FLI) to detect antibodies to FVIII is a sensitive method to identify inhibitors in HA. Evaluations of antibody profiles by various groups have demonstrated that haemophilic inhibitors detected by Nijmegen-Bethesda (NBA) and chromogenic Bethesda (CBA) assays correlate with positivity for anti-FVIII immunoglobulin (Ig) G1 and G4. AIM:This study sought to determine whether FLI could distinguish false-positive FVIII inhibitor results related to LAs from clinically relevant FVIII inhibitors in HA patients. METHODS:Samples from haemophilic and non-haemophilic subjects were tested for LA, specific FVIII inhibitors by NBA and CBA, and anti-FVIII immunoglobulin profiles by FLI. RESULTS:No samples from LA-positive non-haemophilic subjects were positive by FLI for anti-FVIII IgG4. Conversely, 91% of NBA-positive samples from haemophilia subjects were positive for anti-FVIII IgG4. Two of 11 haemophilia subjects had samples negative for anti-FVIII IgG4 and CBA, which likely represented LA rather than FVIII inhibitor presence. CONCLUSIONS:Assessment of anti-FVIII profiles along with the CBA may be useful to distinguish a clinically relevant low-titre FVIII inhibitor from a transient LA in HA patients.

SUBMITTER: Rampersad AG

PROVIDER: S-EPMC6345165 | biostudies-other | 2018 Sep

REPOSITORIES: biostudies-other

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Distinguishing lupus anticoagulants from factor VIII inhibitors in haemophilic and non-haemophilic patients.

Rampersad A G AG Boylan B B Miller C H CH Shapiro A A

Haemophilia : the official journal of the World Federation of Hemophilia 20180713 5

<h4>Introduction</h4>Accurate diagnosis of an inhibitor, a neutralizing antibody to infused factor VIII (FVIII), is essential for appropriate management of haemophilia A (HA). Low-titre inhibitors may be difficult to diagnose due to high rates of false-positive inhibitor results in that range. Transient low-titre inhibitors and false-positive inhibitors may be due to the presence of a lupus anticoagulant (LA) or other non-specific antibodies. Fluorescence immunoassay (FLI) to detect antibodies t ...[more]

PMID: 30004159

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Project description:Hemophilia A is a genetic disorder that results in the deficiency of functional factor VIII protein, which plays a key role in blood coagulation. Currently, the majority of hemophilia A patients are treated with repeated infusions of factor VIII protein. Approximately 30% of severe hemophilia A patients develop neutralizing antibodies to factor VIII (known as factor VIII inhibitors) due to treatment, rendering factor VIII protein infusions ineffective. Previously, mice receiving murine IL-2 complexed with α-murine IL-2 mAbs (JES6-1A12) showed a lack of factor VIII inhibitor formation after factor VIII treatment, which was associated with the proliferation and the activation of factor VIII-specific regulatory T cells (Tregs). In this paper, we evaluated if an Fc-fused mutated protein analog of mouse IL-2, named Fc.Mut24, engineered to selectively promote the expansion of Tregs in vivo can modulate factor VIII-specific immune responses. The mice received one intraperitoneal injection of Fc.Mut24. When the regulatory T cell population reached its highest frequency and peak activation, the mice received a hydrodynamic injection of factor VIII plasmid (day 4) followed by a second Fc.Mut24 dose (day 7). Peripheral blood was collected weekly. Flow cytometry was used to characterize the peripheral blood cell populations, while ELISA and Bethesda assays were used to assess the inhibitor concentrations and the functional titers in plasma. The activated partial thromboplastin time assay was used to assess the functional activities of factor VIII in blood. The mice receiving Fc.Mut24 showed a dramatic and transient increase in the population of activated Tregs after Fc.Mut24 injection. Factor VIII gene therapy via hydrodynamic injection resulted in high anti-factor VIII inhibitor concentrations in control PBS-injected mice, whereas the mice treated with Fc.Mut24 produced no inhibitors. Most significantly, there were no inhibitors generated after a second hydrodynamic injection of factor VIII plasmid administered at 19 weeks after the first injection in Fc.Mut24-treated mice. The mice receiving Fc.Mut24 maintained high levels of factor VIII activity throughout the experiment, while the control mice had the factor VIII activity dropped to undetectable levels a few weeks after the first factor VIII plasmid injection. Our data show that human therapies analogous to Fc.Mut24 could potentially provide a method to prevent inhibitor formation and induce long-term immune tolerance to factor VIII in hemophilia patients.

Project description:Humoral immunity to factor VIII (FVIII) represents a significant challenge for the treatment of patients with hemophilia A. Current paradigms indicate that neutralizing antibodies against FVIII (inhibitors) occur through a classical CD4 T cell, germinal center (GC) dependent process. However, clinical observations suggest that the nature of the immune response to FVIII may differ between patients. While some patients produce persistent low or high inhibitor titers, others generate a transient response. Moreover, FVIII reactive memory B cells are only detectable in some patients with sustained inhibitor titers. The determinants regulating the type of immune response a patient develops, let alone how the immune response differs in these patients remains incompletely understood. One hypothesis is that polymorphisms within immunoregulatory genes alter the underlying immune response to FVIII, and thereby the inhibitor response. Consistent with this, studies report that inhibitor titers to FVIII differ in animals with the same F8 pathogenic variant but completely distinct backgrounds; though, how these genetic disparities affect the immune response to FVIII remains to be investigated. Given this, we sought to mechanistically dissect how genetics impact the underlying immune response to FVIII. In particular, as the risk of producing inhibitors is weakly associated with differences in HLA, we hypothesized that genetic factors other than HLA influence the immune response to FVIII and downstream inhibitor formation. Our data demonstrate that FVIII deficient mice encoding the same MHC and F8 variant produce disparate inhibitor titers, and that the type of inhibitor response formed associates with the ability to generate GCs. Interestingly, the formation of antibodies through a GC or non-GC pathway does not appear to be due to differences in CD4 T cell immunity, as the CD4 T cell response to an immunodominant epitope in FVIII was similar in these mice. These results indicate that genetics can impact the process by which inhibitors develop and may in part explain the apparent propensity of patients to form distinct inhibitor responses. Moreover, these data highlight an underappreciated immunological pathway of humoral immunity to FVIII and lay the groundwork for identification of biomarkers for the development of approaches to tolerize against FVIII.

Dataset Information

Distinguishing lupus anticoagulants from factor VIII inhibitors in haemophilic and non-haemophilic patients.

Publications

Distinguishing lupus anticoagulants from factor VIII inhibitors in haemophilic and non-haemophilic patients.

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