#NIJMEGEN ASSAY
The Bethesda assay was further improved by the Nijmegen modification [241] by buffering the normal pooled plasma with 0.1 M imidazole to pH 7.4 to prevent pH shift during incubation and replacing the buffer as a control with hemophilic or immune-depleted, FVIII-deficient plasma, which prevents dilution of the protein content in the mixture with normal plasma.
The increased specificity without affecting the sensitivity was confirmed in a Canadian study that showed a reduced number of falsely positive assay results with the Nijmegen assay [252].
Hence, the Nijmegen assay is recommended as the standard assay for FVIII inhibitor testing by the International Society of Thrombosis and Hemostasis Factor VIII/IX Scientific Subcommittee.
A schematic representation of the Nijmegen assay is shown in Figure 1. Equal amounts of patient plasma and imidazole-buffered normal pooled plasma, pH 7.4, are incubated for 2 h together with a control mixture of identical normal pooled plasma and FVIII-deficient plasma.
The residual FVIII in the test mixture, defined as the percentage of FVIII remaining in the test mixture relative to the control mixture, is converted to Nijmegen Bethesda units (NBU) by reading from a calibration curve representing a linear correlation between residual FVIII activity (logarithmic) and inhibitor units (linear).
The curve is fully described by 0 and 1 NBU/mL, defined as the amount of inhibitor that results in 100% and 50% residual activity respectively.
The method is only reliable between 0 and 2 NBU/mL and, consequently, samples with elevated titers have to be prediluted, preferably with factor VIII-deficient plasma.
A more sensitive method is warranted in samples with titers below 0.6 NBU/mL and with a suspicion of the presence of an inhibitor because of an abnormal bleeding tendency, low FVIII recovery, or a shortened half-life of infused FVIII.
None of the currently available inhibitor activity assays detects non-neutralizing antibodies, present in 8–10% of hemophilia A patients with or without inhibitors [263, 274].
The influence of non-inhibiting antibodies on hemostasis has not been identified yet, although the formation of such immune complexes may result in increased clearance of these complexes by cells in the innate immune system [285, 296]. Non-neutralizing antibodies can be measured by immunologic methods such as enzyme-linked immunosorbent assay (ELISA), but these methods cannot discriminate between neutralizing and non-neutralizing antibodies.
- Verbruggen B, Novakova I, Wessels H, Boezeman J, van den BM, Mauser- Bunschoten E. The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability. Thromb Haemost. 1995;73(2):247–251. [본문으로]
- Giles AR, Verbruggen B, Rivard GE, Teitel J, Walker I. A detailed comparison of the performance of the standard versus the Nijmegen modification of the Bethesda assay in detecting factor VIII:C inhibitors in the haemophilia A population of Canada. Association of Hemophilia Centre Directors of Canada. Factor VIII/IX Subcommittee of Scientific and Standardization Committee of International Society on Thrombosis and Haemostasis. Thromb Haemost. 1998;79(4):872–875. [본문으로]
- Coutinho A, Kazatchkine MD, Avrameas S. Natural autoantibodies. Curr Opin Immunol. 1995;7(6):812–818. [본문으로]
- Kazatchkine MD, Sultan Y, Burton-Kee EJ, Mowbray JF. Circulating immune complexes containing anti-VIII antibodies in multi-transfused patients with haemophilia A. Clin Exp Immunol. 1980;39(2):315–320. [본문으로]
- Dasgupta S, Navarrete AM, Andre S, et al. Factor VIII bypasses CD91/LRP for endocytosis by dendritic cells leading to T-cell activation. Haematologica. 2008;93(1):83–89. [본문으로]
- Dasgupta S, Navarrete AM, Bayry J, et al. A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes. Proc Natl Acad Sci USA. 2007;104(21):8965–8970. [본문으로]