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. 2016 Oct;14(10):1931-1940.
doi: 10.1111/jth.13438. Epub 2016 Sep 17.

Survey of the anti-factor IX immunoglobulin profiles in patients with hemophilia B using a fluorescence-based immunoassay

B Boylan  1 , A S Rice  2 , A T Neff  3 , M J Manco-Johnson  4 , C L Kempton  5 , C H Miller  2 ; Hemophilia Inhibitor Research Study Investigators
Collaborators, Affiliations

Survey of the anti-factor IX immunoglobulin profiles in patients with hemophilia B using a fluorescence-based immunoassay

B Boylan et al. J Thromb Haemost. 2016 Oct.

Abstract

Essentials Studies characterizing neutralizing antibodies (inhibitors) in hemophilia B (HB) are lacking. The current study describes anti-factor (F) IX antibody profiles in 37 patients who have HB. Anti-FIX IgG4 levels exhibited a strong positive correlation with Nijmegen-Bethesda results. These data will help to more clearly define, predict, and treat alloantibody formation in HB.

Summary: Background Hemophilia B (HB) is an inherited bleeding disorder caused by the absence or dysfunction of coagulation factor IX (FIX). A subset of patients who have HB develop neutralizing alloantibodies (inhibitors) against FIX after infusion therapy. HB prevalence and the proportion of patients who develop inhibitors are much lower than those for hemophilia A (HA), which makes studies of inhibitors in patients with HB challenging due to the limited availability of samples. As a result, there is a knowledge gap regarding HB inhibitors. Objective Evaluate the largest group of patients with inhibitor-positive HB studied to date to assess the relationship between anti-FIX antibody profiles and inhibitor formation. Methods A fluorescence immunoassay was used to detect anti-FIX antibodies in plasma samples from 37 patients with HB. Results Assessments of antibody profiles showed that anti-FIX IgG1-4 , IgA, and IgE were detected significantly more often in patients with a positive Nijmegen-Bethesda assay (NBA). All NBA-positive samples were positive for IgG4 . Anti-FIX IgG4 demonstrated a strong correlation with the NBA, while correlations were significant, yet more moderate, for anti-FIX IgG1-2 and IgA. Conclusions The anti-FIX antibody profile in HB patients who develop inhibitors is diverse and correlates well with the NBA across immunoglobulin (sub)class, and anti-FIX IgG4 is particularly relevant to functional inhibition. The anti-FIX fluorescence immunoassay may serve as a useful tool to confirm the presence of antibodies in patients who have low positive NBA results and to more clearly define, predict, and treat alloantibody formation against FIX.

Keywords: factor IX; factor IX deficiency; hemophilia B; immunoassay; inherited blood coagulation disorders.

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Conflict of interest statement

of Conflict of Interests C. Kempton reports grants from CDC Foundation during the conduct of the study; grants and personal fees from Novo Nordisk, as well as personal fees from Baxalta, Biogen Idec, and CSL Behring outside the submitted work. A. Neff reports grants from CDC Foundation during the conduct of the study; grants from Baxter, Novo Nordisk, and CSL Behring, as well as personal fees from Alexion outside the submitted work. C. Miller reports grants from CDC Foundation during the conduct of the study. A. Dunn reports grants and personal fees from Biogen, Bayer, Baxalta, and CSL Behring; grants from Octopharma, Kedrion, and Novo Nordisk outside the submitted work. S. R. Lentz reports grants from CDC Foundation during the conduct of the study; personal fees from Novo Nordisk outside the submitted work. M. Tarantino reports personal fees and other from Bleeding and Clotting Disorders Institute, Novo Nordisk, Baxalta, Grifols, Amgem, Pfizer, Biogen, HRSA, and CDC Foundation outside the submitted work. A. Escobar reports grants and personal fees from Pfizer, personal fees from Baxalta, Novo Nordisk, Bayer, and CSL Behring outside the submitted work. C. Lessinger reports grants from Baxalta, CSL Behring, and Roche; advisory board participation in Baxalta, Bayer, CSL Behring, Kedrion, Novo Nordisk, Roche, Pfizer, Biogen, and LFB. J. C. Gill reports grants from Centers for Disease Control and Prevention during the conduct of the study; grants and personal fees from CSL Behring Shire and Bayer outside the submitted work. B. M. Wicklund reports grants from CDC Foundation during the conduct of the study; personal fees from Novo Nordisk, Biogen, Baxalta-Shire, Bayer, and Oakstone-EBIX outside the submitted work.

Figures

Figure 1
Figure 1. Demonstration of Anti-FIX fluorescence immunoassay sensitivity and specificity
Histograms represent median fluorescence intensities obtained on plasma samples diluted 1:30000, 1:3000, or 1:30. 1:30 dilutions were pre-incubated +/− 300μg/ml recombinant FIX. Dashed lines represent the thresholds of positivity.
Figure 2
Figure 2. Fluorescence immunoassay results for anti-FIX antibodies in plasma from patients who have hemophilia B (HB) and healthy controls
Data points represent results for individual plasma samples for IgG1 (A), IgG2 (B), IgG3 (C), IgG4 (D), IgA (E), and IgE (F). Results are displayed on a log scale for control plasmas from healthy donors and patients with hemophilia B who tested negative (< 0.3) or positive (≥ 0.3 NBU) by the Nijmegen-Bethesda assay. Thresholds for positivity, which, are set at two standard deviations above the mean MFI of control samples and represented by dashed lines are 13.6, 15.0, 15.4, 9.8, 600.5, and 24.6 for IgG1, IgG2, IgG3, IgG4, IgA, and IgE, respectively. * P ≤ 0.0001; **P ≤ 0.002; *** P = 0.0375
Figure 3
Figure 3. Correlation of anti-FIX fluorescence immunoassay and Nijmegen-Bethesda assay results for samples from patients with hemophilia B
As shown, individual data points represent results obtained on hemophilia B patient samples using the immunoglobulin-specific FLIs (IgG1 (A), IgG2 (B), IgG3 (C), IgG4 (D), IgA (E), or IgE (F)) plotted on a log scale against NBA results for the same plasma sample. Samples positive for one or both assays (filled circles) were used to calculate linear correlations according to Spearman (r). Dashed lines represent the thresholds of positivity.
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