Laboratory testing for factor inhibitors

Abstract

Summary. Inhibitor assays are performed when patients present with unexplained prolonged routine coagulation test times and unexpected and/or unusual bleeding (potential for acquired haemophilia) as well as being a part of normal congenital haemophilia management and monitoring, particularly when bleeding occurs on therapy, or when increments in factor levels post-factor replacement remain lower than expected. In this article, we will describe the assays used, as well as their development, pitfalls in testing such as inter-laboratory variability and false negative/ positive results, as well as some strategies for overcoming these pitfalls and potential alternative test approaches. The inter-laboratory coefficient of variation often approaches (and sometimes exceeds) 50%, as evidenced by various external quality assessment groups, and this variability has not improved over recent years. Additional important considerations include appropriate interpretation of test results, repeat testing for confirmation, and assessment of recovery as part of the diagnostic process.

Heat treatment of samples improve the performance of the Nijmegen–Bethesda assay in hemophilia A patients undergoing immune tolerance induction

“…Another important scenario is related to high coefficient of variation (CV %) of Bethesda assay and of the Nijmegen-Bethesda assay. The poor reproducibility of the test is even more pronounced in inhibitor titers below 20 BU [10]. The lack of sensitivity mainly in low titer inhibitor range can interfere in different clinical settings, such as the choices of the treatment product and in management of ITI therapy, resulting in less effective replacement therapy and increasing bleeding complications [10].…”

“…The poor reproducibility of the test is even more pronounced in inhibitor titers below 20 BU [10]. The lack of sensitivity mainly in low titer inhibitor range can interfere in different clinical settings, such as the choices of the treatment product and in management of ITI therapy, resulting in less effective replacement therapy and increasing bleeding complications [10]. In other words, no inhibitor detection by Nijmegen-Bethesda assay due to high variability results inter-laboratory can also affect treatment choices.…”

Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review

“…Coefficients of variation (CVs) as high as 50% and false-positive rates up to 32% have been seen on distributed specimens. 88,89 It is likely that differences in methods and reagents contribute to this variability among well-qualified laboratories. 88 Among North American laboratories surveyed by the North American Specialized Coagulation Laboratory Association, 20% reported using the Nijmegen method, 10% the Bethesda assay and 70% a combination of components best described as a hybrid assay.…”

Reduction of Dilution Error in Elisas Using an Internal Standard

“…Among assay-procedure based errors, positioning [9][10][11] effects are a particular problem in microtiter plate assays [12][13][14][15]. Positioning effects can be linked to a number of sources such as plate treatment, reader effects, temperature gradients and/or multichannel equipment errors including pipets, liquid handlers and washers.…”

“…Sample-associated error factors include tube adsorption during dilution steps, sample matrix effects in particular from ex vivo samples or sample stability depending on the nature of the antigen and the antibodies used [9][10][11]. These sources of variability are assay-specific and must be addressed on a case-by-case basis.…”