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

Abstract

Inhibitors are antibodies directed against haemophilia treatment products which interfere with their function. Factor VIII (FVIII) inhibitors in haemophilia A and factor IX (FIX) inhibitors in haemophilia B are significant clinically when they require a change in a patient’s treatment regimen. Their persistence may increase morbidity and mortality. Multiple laboratory tests are now available for detecting and understanding inhibitors in haemophilia. Inhibitors are traditionally measured by their interference in clotting or chromogenic factor assays. They may also be detected using immunologic assays, such as enzyme-linked immunosorbent assay or fluorescence immunoassay. Anti-FVIII or anti-FIX antibodies of IgG4 subclass best correlate with the presence of functional inhibitors. Improvements in inhibitor measurement have been recently introduced. Preanalytical heat treatment of patient specimens allows testing of patients without delaying treatment. Use of chromogenic and immunologic assays may aid in identification of false-positive results, which are frequent among low-titre inhibitors. Validated reagent substitutions can be used to reduce assay cost. New methods for defining assay positivity and reporting low-titre inhibitors have been suggested. Challenges remain in the areas of quality control, assay standardization, monitoring of patients undergoing immune tolerance induction therapy and testing in the presence of modified and novel treatment products.

Safety and efficacy of turoctocog alfa in the prevention and treatment of bleeds in previously untreated paediatric patients with severe haemophilia A: Results from the guardian 4 multinational clinical trial

“…Therefore, it is important to consider differences in study design, sample size and inhibitor‐testing methodologies when comparing PUP studies. Multiple laboratory tests are available for detecting inhibitors in haemophilia, and new methods for defining assay positivity have been suggested; however, there are still ongoing challenges in the areas of assay standardization and testing in the presence of modified and novel treatment products …”

Laboratory coagulation tests and emicizumab treatment A United Kingdom Haemophilia Centre Doctors' Organisation guideline

“…12 Chromogenic assays for coagulation factors or treatments other than FVIII. Pre-analytical heat inactivation methods, routinely used to inactivate residual FVIII prior to performing the Bethesda assay, will not completely remove emicizumab, an IgG4 bispecific antibody.…”

“…Pre-analytical heat inactivation methods, routinely used to inactivate residual FVIII prior to performing the Bethesda assay, will not completely remove emicizumab, an IgG4 bispecific antibody. 12 Chromogenic assays for coagulation factors or treatments other than FVIII.…”

Reagent substitution in the chromogenic Bethesda assay for factor VIII inhibitors

“…1 Modification of the traditional NBA 2 to use a chromogenic measurement of FVIII as the endpoint is necessary for measurement of FVIII inhibitors in the presence of heparin, lupus anticoagulants, or by-passing agents such as emicizumab, due to their interference in clot-based assays. [3][4][5] Parallel testing has shown this modification to produce similar results to the NBA in the absence of these interfering substances. 6 In the clot-based NBA, substitution of imidazolebuffered bovine serum albumin (IB-BSA) for FVIII-deficient plasma (FVIIIDP) as diluent in control mixtures and specimen dilutions has been shown to produce equivalent results when the threshold for positivity was slightly adjusted.…”