Comparison of clot-based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study

Abstract

Background: Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety and assessment of population trends.

Methods: Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA) and a novel fluorescence immunoassay (FLI).

Results: NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU) < 0.5 and positive on 42/42 specimens (100%) with NBU ≥ 2.0 and 43/80 specimens (53.8%) with NBU 0.5-1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0-1.9 NBU specimens and 43.1% and 50.0% of 0.5-0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P = 0.004). Among 21 new inhibitors detected by NBA, five (23.8%) with 0.7-1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5-1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%).

Conclusions: FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5-1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays.

Keywords: factor VIII; factor VIII inhibitor; hemophilia A; immunology and fluorescence immunoassay.

Figure 1

Correlation of Nijmegen-Bethesda Units (NBU) and Chromogenic Bethesda Units (CBU) in specimens with ≥2.0 NBU. Two points were outside the range of the graph at NBU 207.6, CBU 108.8 and NBU 688.0, CBU 512.0.

Figure 2

Results of fluorescence immunoassay in fluorescence immunoassay units (FLIU) in normal individuals (n=83) and hemophilia patients with negative Nijmegen-Bethesda assay results (n=169).

Figure 3

Comparison of positivity rates of Chromogenic Bethesda Assay and Fluorescence Immunoassay (FLI) with Nijmegen-Bethesda Units. A. All specimens. B. Specimens with NBU<1.0.

Figure 4

Dilution curves for inhibitors of 13.3 Nijmegen-Bethesda units (NBU) (A), 30.8 NBU (B), and 48.5 NBU (C). Solid lines represent the Nijmegen-Bethesda Assay. Dashed lines represent the Chromogenic Bethesda Assay with the cutoff for positivity (dotted line) at 70% residual activity (0.5 NBU). D. Dilution curve of factor VIII (FVIII) activity assay. Solid line represents FVIII clotting assay. Dashed line represents FVIII chromogenic assay.

Figure 5

Dilution curves of the fluorescence immunoassay. The dotted line represents the cutoff for assay positivity at fluorescence immunoassay units (FLIU) =0.466.

Figure 6

Nijmegen-Bethesda Assay (NBA) dilution curves of inhibitor plasmas. A: Concordant specimens [positive NBA and positive Chromogenic Bethesda Assay (CBA)].B: Discordant specimens (positive NBA and negative CBA). Dashed lines at 75% and 25% residual factor VIII activity define the interval within which NBU are calculated, representing the 0.4 and 2.0 NBU levels, respectively.