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SPLTRAK Abstract Submission
Validation of Chromogenic Factor VIII Activity Assays at Children’s Hospital Los Angeles (CHLA)
Joshua M. Brown, PhD1, Melissa Arevalo1, Desiree Tan-Castillo1, Edward Leung, PhD, DABCC, FAACC1,2, Guy Young, MD1,2, Maurice O'Gorman, MBA, MSc, PhD, D(ABMLI)1,2
1Children's Hospital Los Angeles, Los Angeles, CA, United States/2University of Southern California Keck School of Medicine, Los Angeles, CA, United States

Introduction: Therapies designed to prevent bleeding may interfere with traditional methods of evaluating coagulation hemostasis, necessitating the adoption of new methods or modification of existing methods to assess factor activity. The novel therapeutic bispecific antibody, emicizumab, dramatically improves the treatment for hemophilia A patients by effectively replacing the role of factor VIII; however, it has the unintended consequence of interfering with APTT-based assays. Unlike the one stage clotting assays, chromogenic assays using bovine-derived reagents are unaffected by the treatment with humanized emicizumab allowing for the measurement of endogenous and exogenous factor VIII and well as inhibitors via a modified Bethesda assay. On the other hand, chromogenic assay using human-derived reagents allows for the pharmacodynamic assessment of emicizumab, allowing for an estimation of drug levels. The development of these two methods has allowed our physicians to select the most appropriate test to monitor patients being treated with emicizumab.  Our validation of these chromogenic assays will be presented here.  Methods: Validation studies were performed for bovine- and human-based FVIII chromogenic assays (Chromogenix: Instrumentation Laboratory, 180 Hartwell Road, Bedford, MA 01730-2443; Aniara Diagnostica: 155 Rue d’Eragny, 95000 Neuville-sur-Oise, France). Each assay was analyzed for precision, accuracy, linearity, and verification of the reference interval. Precision was measured using 3 sets of controls for the bovine-based method (n=10), and 4 sets of controls for the human-based method (n=18). Assay linearity was assessed using cryoprecipitate and Universal Reference Plasma (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA 02451). For accuracy assessment, results from bovine- or human-based chromogenic methods were compared to results determined using the clot method (n=41 and n=26, respectively). The reference interval was determined by performing the assay on 23 normal donor specimens (12 female, 11 male) for the bovine-based assay, and 20 normal donor specimens (10 female, 10 male) for the human-based assay. Results: Both the bovine- and human-based FVIII chromogenic assays had satisfactory precision parameters, i.e.  CVs of <15%. The assays were also found to be linear (R2 >95%) up to 331% for the human-based method and up to 242% for the bovine-based method. Both chromogenic assays gave results that compared well with results determined from the clot-based assays, with overall biases of -7.6% for the bovine chromogenic method and 5.6% for the human chromogenic method. Testing of normal patient samples allowed the establishment of a reference interval of 50 – 150% factor VIII activity for both chromogenic methods. Conclusion: Laboratories must ensure that reported clotting factor results accurately represent the physiological state of the patient and are not affected by new and novel therapies. Recently FDA approved therapeutics like emicizumab interfere with traditional assays and may provide misleading results. The validation of chromogenic factor VIII assays at CHLA allows physicians to monitor factor activity, inhibitor levels as well as activity levels of emicizumab in hemophilia patients.