Revealing and IgG4 analysis to factor VIII in haemophilia-A patients with and without inhibitors

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Highlights

  • The acquisition of factor VIII inhibitors poses major management challenges for Hemophilia A (HA) patients.

  • Most (Factor VIII) inhibitors are immunoglobulin G4 (IgG4) and G1 (IgG1) subclasses, with IgG4 being the most prevalent.

  • The ELISA-based screening proved to be more viable in a resource-constrained scenario.

  • At cutoff OD of 0.502, in-house ELISA sensitivity was 93.3%, % specificity 97.0, NPV 97% and PPV 93.3%, respectively.

  • IgG4 ELISA is an effective method for detecting neutralizing or functionally significant FVIII inhibitors.

Abstract

Introduction

The acquisition of factor VIII inhibitors poses major management challenges for haemophilia A (HA) patients. Most (Factor VIII) inhibitors are immunoglobulin G4 (IgG4) and G1 (IgG1) subclasses, with IgG4 being the most prevalent. The Nijmegen Bethesda Assay (NBA) was used to quantify inhibitors. However, the requirement for a large sample volume, accompanying costs, and required technical expertise complicate NBA, particularly in developing countries. ELISA-based screening proved to be more viable in a resource-constrained scenario.

Aim

This study aimed to standardise and evaluate an in-house IgG4 ELISA for the detection of haemophilia A inhibitors.

Methods

This study enrolled thirty HA patients with inhibitors, thirty three HA without inhibitors, and 33 healthy controls. Standardisation of in-house IgG4 ELISA was performed. The checkerboard method was employed to optimise plasma-derived Factor VIII concentrations (HEMOFIL M Baxalta US Inc.), sample dilutions, and anti-human IgG4-HRP conjugate (Southern Biotechnology, USA). The samples were evaluated three times, and the mean optical density (OD) was used to determine the cutoff.

Results

Using a cutoff OD (mean±2SD) of 0.502 in our in-house ELISA, we could differentiate healthy controls and HA without inhibitors from HA with inhibitors with 93.3 % sensitivity, 97.0 % specificity, 97 % NPV, and 93.3 % PPV, respectively. However, the accuracy was 95.83 %. The two-way mixed-effects model, interclass correlation (ICC) derived by Cronbach’s Alpha was 0.912 (p = 0.001) and close to perfect agreement.

Conclusions

IgG4 ELISA is an effective method for detecting neutralising or functionally significant FVIII inhibitors, particularly in resource-constrained settings, following which patients may be referred to referral laboratories for quantification of inhibitors.

Introduction

According to data from the World Federation of haemophilia (WFH) 2016 Annual Global Survey Report, India has the highest number of haemophilia populations with 18,353, of which 15,218 had haemophilia A (HA), 2,379 had haemophilia B (HB), and 756 had an unexplained category [1].

The therapy includes replacement therapy with plasma-derived or recombinant Factor VIII (FVIII). The development of Antibodies against FVIII is a major health concern in patients with HA. These inhibitory antibodies bind to the infused FVIII, reducing its half-life and suppressing its coagulant ability. This leads to bleeding episodes becoming superior to standard treatment. In contrast, possible treatment methods such as inhibitor bypassing drugs (inhibitors) are required.

The cumulative incidence of inhibitor development is between 20%–30% in severe HA and between 5 %–10 % in moderate or mild disease [2]. The global population of patients with HA who are clinically diagnosed with inhibitors is approximately 4711 [1].

In patients with HA, the antibodies to exogenous FVIII are polyclonal immunoglobulin G (IgG) antibodies, with the majority belonging to the IgG4 subtype. Further IgG1 and IgG2 subtypes are present, but they do not bind to complement. Anti-FVIII IgG4 antibodies correlate best with the presence of functional inhibitors [[3], [4], [5]].

Typically, inhibitors are quantified by their interference with clotting or chromogenic factor assays. Moreover, they can be detected using immunologic assays such as enzyme-linked immunosorbent assays or fluorescence immunoassays.

In clinical practice, functional antibody responses to FVIII are commonly used to identify FVIII inhibitors using the Classical Bethesda Assay (CBA) or the Nijmegen-modified Bethesda Assay (NBA). The CBA, which quantifies FVIII inhibitor plasma concentrations, is the accepted gold standard for detecting FVIII inhibitors in HA. However, this technique adequately detects low inhibitor titers, yields false-negative outcomes, and samples with large inhibitor titers require repeated assays following dilution. CBA and NBA are technically intensive assays that require highly skilled personnel and a large sample volume. They also consume a significant amount of chemicals, making them expensive. This is a significant impediment to inhibitor detection in developing countries.

Previous studies employing ELISA to detect FVIII inhibitors and their immunoglobulin subclass revealed an acceptable or higher sensitivity [6] and a strong correlation between CBA and ELISA [7].

The predominant IgG subclasses in patients treated with FVIII inhibitors are IgG4 and IgG1 [5,8]. IgG4 was absent in patients who did not receive FVIII inhibitors or healthy controls [8]. These findings are promising since they demonstrate a specific immune regulatory process for the development of FVIII-specific IgG4 and the synthesis of FVIII inhibitors. Measuring this subset of antibodies (ideally IgG4 and IgG1) in everyday clinical practice may be highly beneficial for discovering clinically significant antibodies. In comparison to NBA, ELISA enables quick screening that may be accessible before Bethesda confirmation.

The purpose of this study was to standardise and evaluate an in-house indirect IgG4 ELISA for screening and to diagnose FVIII inhibitors and to compare it to the Nijmegen-Bethesda assay in haemophilia-A patients.

Section snippets

Materials and methods

The research group consists of a subset of 300 cases identified through camps held in various cities throughout North India in collaboration with the haemophilia Federation of India and through direct referral to our laboratory. All HA, with or without inhibitor, were severe haemophilia A patients with less than 1 % Factor VIII bioactivity. The study categories are listed in Table 1, along with their respective inclusion and exclusion criteria. This study was a case series comprising three

Results

Study group distribution with clinical characteristics and inhibitor titers are given in Table 2.

Discussion

Inhibitors of coagulation factors are mainly divided into neutralising and non-neutralising inhibitory antibodies. Inhibitors can either block the action of the factor or lead to their clearance from the circulation after combining it. This, in effect, leads to ‘neutralisation’ of clotting factor activity and is known as a functional inhibitor. Anti-FVIII or anti-FIX antibodies that do not interfere with clotting factor function may also be present; these have been called “non-neutralising” or

Conclusion

IgG4 ELISA for detecting neutralising or functional FVIII inhibitors may prove to be a valuable technique for detecting clinically significant inhibitors, especially in resource-poor setup, following which patients may be referred for quantification of inhibitors to referral laboratories. This approach may benefit the haemophilia treatment centres as they continue to struggle for inhibitor quantification.

Data availability

Data will be made available on request.

Funding

Study group was part of a cohort of haemophilia A cases for inhibitor testing (Reference 9) funded by the Department of Science and Technology (DST), New Delhi, India. Hence, the current study may be considered partially funded by DST.

Ethics

Institutional Ethics Committee, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, UP, India (Ref: IEC 33/16).

Contributorship statement

NPA: Data collection, data analysis, manuscript preparation and revision.

KR- Sample collection, history & data collection of enrolled patients.

VT- Conceptualisation, Standardizing in-house ELISA, validation and diagnostic evaluation of in-house IgG4 ELISA, manuscript preparation and editing.

SA- Sample collection, data collection, data analysis.

NH-Conceptualization, data analysis, manuscript revision.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

Authors wish to acknowledge Department of Science and Technology (DST), New Delhi, India for providing SERB grant support (Ref. no SB/SO/HS/025/2014) as partial support for this research.

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