Abstract Number: PB0563
Meeting: ISTH 2022 Congress
Theme: Coagulation and Natural Anticoagulants » Coagulation Factors and Inhibitors
Background: Treatment of hemophilia A (HA) with factor VIII (FVIII) replacement therapy can be complicated by inhibitor formation against exogenous infused FVIII, rendering therapy ineffective. Inhibitors can also be formed against endogenous FVIII, causing acquired HA (AHA). In both cases inhibitor formation is related with an increased bleeding risk. The ISTH recommends the Nijmegen Bethesda Assay (NBA) for detecting and quantifying inhibitors, but the assay is only able to detect inhibitor levels above the cut-off level of 0.6 NBU/ml.
Aims: Introduction of the ultra-sensitive Nijmegen Inhibitor Assay (usNIA) for detecting very low-titer FVIII inhibitors.
Methods: The usNIA is an adaptation of the NBA, in which the ratio of patient sample to normal pooled plasma is changed from 1:1 (NBA) to 9:1 (usNIA). Residual FVIII activity was determined with a chromogenic assay (FVIII:C, Biophen Biomed) using a low FVIII:C calibration line (0-15%) and inhibitor titers were extrapolated. A recombinant FVIII inhibitor with a known titer was diluted (0.05-0.80 NBU/mL) to generate a calibration curve and assess the usNIA lower limit of quantification (LLOQ, figure). Furthermore, corresponding usNIA, NBA and FVIII activity levels were determined in sequential plasma samples from three patients with AHA to analyze the interplay between these parameters. Patients gave consent for research usage of left-over diagnostic material
Results: Healthy controls (n=49) had no detectable inhibitors (mean residual FVIII 109%, range 102-116%). The usNIA LLOQ is determined as 0.1 NBU/mL, with an associated coefficient of variation of 12%. In AHA patients the usNIA detected FVIII inhibitory antibodies, even when the NBA indicated an undetectable titer ( < 0.6 NBU/mL, table).
Conclusion(s): The usNIA is able to detect low-titer FVIII inhibitor titers up to 0.1 NBU/ml. The usNIA will be further validated in clinical practice to prove its value in early inhibitor detection and therapy adjustments in patients with congenital and acquired HA.
Figure: Calibration line of the ultra-sensitive Nijmegen Inhibitor Assay -usNIA-.
The dots represent the residual FVIII activity level measured in different dilutions of a recombinant FVIII inhibitor with a known titer. The fitted logarithmic line was used to calculate the usNIA titers in the patient samples.
Table: Course of inhibitor titers of patients with acquired hemophilia A.
Neg indicates a negative inhibitor titer -i.e. under the Lower Limit of Quantification of the assay, which is set at 0.6 NBU/mL for the NBA, and at 0.1 NBU/mL for the usNIA-.
FVIII: factor VIII; NA: not available; NBA: Nijmegen Bethesda Assay; NBU: Nijmegen Bethesda Units; usNIA: ultra-sensitive Nijmegen Inhibitor Assay.
To cite this abstract in AMA style:
Valke L, Verhagen M, Mulders B, Polenewen R, Schols S, van Heerde W, Meijer D. The ultra-sensitive Nijmegen Inhibitor Assay detects very low-titer factor VIII inhibitors. [abstract]. https://abstracts.isth.org/abstract/the-ultra-sensitive-nijmegen-inhibitor-assay-detects-very-low-titer-factor-viii-inhibitors/. Accessed October 1, 2023.« Back to ISTH 2022 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/the-ultra-sensitive-nijmegen-inhibitor-assay-detects-very-low-titer-factor-viii-inhibitors/