LAUSR

Dear Editors: Development of antibodies interfering with the function of factor VIII (FVIII) replacement products is one of the most significant complications in the treatment of hemophilia A (HA). Laboratory testing for such antibodies, called inhibitors, is an important part of hemophilia care and is conducted both to identify the cause of treatment failure and as routine screening to detect early antibody appearance. Treatment of HA patients who develop inhibitors is often carried out by giving repeated doses of FVIII to induce immune tolerance and allow use of FVIII or by use of by-passing agents that act by facilitating coagulation without the need for FVIII. The newest by-passing product, emicizumab (Hemlibra®), is a bispecific antibody that mimics the function of FVIII by bringing factor IXa and factor X (FX) together to produce the Xa complex.1,2 Emicizumab, which is long acting and given subcutaneously, is now widely available for use in patients both with and without inhibitors to avoid frequent use of intravenous FVIII replacement. Monitoring of patients treated with emicizumab presents challenges for the clinical laboratory, because the drug interferes with the FVIII one-stage assay (OSA).1-3 Emicizumab reacts in a FVIII chromogenic substrate assay (CSA) only if human-derived FX is used; it does not react with the bovine FX used in some FVIII CSA.3 This differential species reactivity has been exploited to allow measurement of FVIII and FVIII inhibitors in the presence of emicizumab by using such a CSA.4 The traditional assays for FVIII inhibitors based on a OSA include a modified Nijmegen-Bethesda assay (NBA) that uses preanalytical heat treatment of patient plasma to remove infused or endogenous FVIII without destroying the antibodies to be measured5; however, emicizumab, itself an antibody, cannot be removed by this method.1,2 We6 and others (as reviewed in Miller7) have previously demonstrated improved performance of a chromogenic Bethesda assay (CBA) over the NBA due to its insensitivity to nonspecific inhibitors of coagulation in patients treated with FVIII products. The aim of this study was to describe the performance characteristics of the CBA for FVIII inhibitor measurement in patients receiving emicizumab. Data were analyzed from 800 specimens collected from subjects with congenital HA enrolled in the Registry for Bleeding Disorders Surveillance of the Community Counts program8 at US Hemophilia Treatment Centers who were receiving emicizumab as their primary treatment product. Participants were not required to give informed consent for surveillance specimens. Data on previous inhibitor history collected from the enrolling sites on forms submitted with each specimen were available for 781 specimens from 648 patients. Specimens were collected as previously described5 into 3.2% sodium citrate in a ratio of 1:9 with blood and centrifuged at 1600 × g for 20 minutes at 4°C, followed by a second centrifugation of separated plasma under the same conditions in polypropylene tubes. The separated plasma was shipped to CDC overnight on cold packs or frozen on dry ice and there aliquoted and stored in polypropylene tubes at −70°C. The chromogenic Bethesda assay (CBA) was performed as previously described,6 including heating of patient plasma to 56°C for 30 minutes and centrifugation prior to testing. One chromogenic Bethesda unit (CBU) was defined as the amount of inhibitor per milliliter (mL) of patient plasma which inactivates 50% of the FVIII activity in 1 mL of pooled normal plasma (PNP) during a 2-hour incubation at 37°C. FVIII activity was measured using a CSA (Siemens Factor VIII Chromogenic Assay, Siemens, Marburg, Germany) performed on a STAR Evolution (Diagnostica Stago). Plasma diluted 1:31 in imidazole buffer (Siemens) was incubated with bovine FX, bovine factor IXa, bovine thrombin, CaCl2, and phospholipid for 90 seconds at 37°C. Factor Xa substrate with a thrombin inhibitor and a stopping buffer was added. The change in absorbance per minute was read at 405 nanometers. Antibodies binding to FVIII were measured by fluorescence immunoassay, as previously described.9 Results were expressed as median fluorescence intensity (MFI). The threshold for positivity was set at two standard deviations above the mean MFI of the results obtained for healthy subjects. Group frequencies were compared by Fisher's exact test with significance set at P < .05 using GraphPad Prism 8.3 (GraphPad Software Inc). The threshold for positivity for the CBA was evaluated by two methods previously used for the NBA: examination of CBA results in patients with and without history of inhibitor5 and comparison of CBA results with the presence of anti-FVIII IgG4 antibodies.10 Anti-FVIII antibodies of IgG4 subclass have been shown to be most closely linked to the presence of a functional inhibitor, other subclasses also appearing in inhibitor-negative patients.9,11