PB0563 - The ultra-sensitive Nijmegen Inhibitor Assay detects very low-titer factor VIII inhibitors.

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Lars Valke, MD

PhD candidate
Radboud University Medical Center, Nijmegen, the Netherlands; Hemophilia Treatment Center, Nijmegen-Eindhoven-Maastricht, the Netherlands
Nijmegen, Gelderland, Netherlands

Co-Author(s)

Marieke Verhagen, MD

PhD student, MD
Department of Hematology, Radboud University Medical Center, Nijmegen, the Netherlands. Hemophilia Treatment Center Nijmegen-Eindhoven-Maastricht, Nijmegen, the Netherlands. Laboratory of hematology, Radboud university medical center, Nijmegen, the Netherlands
Nijmegen, Gelderland, Netherlands
BM

Bart Mulders

Laboratory technician
Department of Laboratory Medicine, Radboud university medical center, Nijmegen, the Netherlands
Nijmegen, Gelderland, Netherlands
RP

Robert Polenewen

Laboratory technician
Department of Laboratory Medicine, Radboud university medical center, Nijmegen, the Netherlands
Nijmegen, Gelderland, Netherlands
SS

Saskia E.M. Schols

Internist-hematologist
Department of Hematology, Radboud university medical center, Nijmegen, The Netherlands. Hemophilia Treatment Center Nijmegen – Eindhoven – Maastricht, The Netherlands
Nijmegen, Gelderland, Netherlands
Wv

Waander L. van Heerde

Biochemist, Coagulation Physiologist, Chief Scientific Officer Enzyre BV
Department of Hematology, Radboud university medical center, Nijmegen, The Netherlands. Hemophilia Treatment Center Nijmegen – Eindhoven – Maastricht, The Netherlands. Enzyre BV, Novio Tech Campus, Nijmegen, The Netherlands.
Nijmegen, Gelderland, Netherlands
DM

Daniëlle Meijer

Clinical chemist
Department of Laboratory Medicine, Laboratory of Hematology, Radboud university medical center, Nijmegen, the Netherlands
Nijmegen, Gelderland, Netherlands

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.