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Introduction: Non‐factor replacement therapies are emerging as prophylactic treatment options in haemophilia A or B (HA/HB) with and without inhibitors. Concizumab is an anti‐tissue factor pathway inhibitor (TFPI) monoclonal antibody preventing factor (F)Xa inhibition and enhancing thrombin generation. Based on experience with other non‐factor therapies and extended half‐life products, there is a focus on potential interference with common clinical coagulation assays used to monitor patients treated with concizumab. Aim: To evaluate the impact of concizumab on standard clinical coagulation assays. Methods: Plasma samples (normal, HA/HB with/without inhibitors) in the presence/absence of added concizumab (250–16,000 ng/mL) were analysed in clinical assays including activated partial thromboplastin time (aPTT), prothrombin time (PT), FVIII and FIX one‐stage clot and chromogenic substrate assay, assays for detecting FVIII or FIX inhibitors and other assays for coagulation factors. Results: Concizumab did not impact PT assays, but resulted in a small shortening of aPTT (up to 5 s in haemophilia plasma and 0.4 s in normal plasma). Concizumab had no, or only a minor impact on FVIII and FIX activity assays or Bethesda inhibitor assays. FXI and FXII activity in normal plasma, as measured by single factor aPTT‐based assay, was significantly increased in the presence of concizumab (+11% each). This was also the case for FVII and FX measured by PT‐based assays using plasma with 25% of FVII or FX (+64% and +22%, respectively). Conclusion: The presence of concizumab did not, or only slightly, influence the outcome of standard clinical coagulation assays relevant for HA and HB. [ABSTRACT FROM AUTHOR]

Key Clinical Message: Acquired inhibitors of blood coagulation are rare but of clinical importance. Prothrombin is a vitamin K‐dependent protein, and acquired antibodies toward prothrombin are often associated with the presence of lupus anticoagulant. We describe a previously healthy 70‐year‐old man presenting with both hemorrhage and thrombosis as well as a prolonged prothrombin time. At arrival at the hospital, he was diagnosed with deep venous thrombosis, and an enlarged lymph node in the left groin was noted (revealed as follicular lymphoma grade 1 by biopsy). Prothrombin activity and antibody titer were followed for 5 months with 15 sampling time points to monitor the treatment outcome of the patient. Diagnostic work‐up identified prothrombin deficiency as cause of bleeding. A nonneutralizing calcium‐dependent antiprothrombin antibody was found, suspected to increase the clearance of prothrombin, which has previously only occasionally been reported. Lupus anticoagulant was ruled out and thrombosis was judged to be caused by a combination of malignant disease and stagnant venous flow following enlarged lymph nodes in the groin. This report illustrates how investigation of prolonged global coagulation tests, triggered the diagnosis of a rare but critical condition, immune‐mediated prothrombin deficiency. The diagnosis is challenging and involves proper differential diagnosis. [ABSTRACT FROM AUTHOR]

Introduction: The development of neutralising (inhibitors) and non‐neutralising antibodies (NNAs) is a complication to factor replacement therapy in haemophilia. The diagnostic methods available lack standardisation, have high inter‐laboratory variation, and false‐negative as well as false‐positive results may affect treatment. Both functional inhibitors and NNAs may be detected with higher reproducibility, sensitivity and specificity using the immunological Luminex xMAP‐based fluorescence‐immunoassay (xFLI). Aim: Validation of our xFLI and comparability with enzyme‐linked immunosorbent assay (ELISA) and chromogenic Nijmegen‐Bethesda assay (CBA) for anti‐FVIII antibodies in haemophilia A (HA) patients. Methods: The xFLI method was developed with full‐length and B‐domain deleted factor coupled to magnetic beads, optimised and validated for performance characteristics. Comparability with ELISA and CBA was evaluated in HA patient samples (n = 112), serial samples in six inhibitor patients and reference interval and decision‐limits in healthy donors (n = 44). Results: The intra‐ and inter‐assay precision (CV%) for the xFLI method was below 6% and detection limit (LLOQ).084 ng/mL (NovoEight). All ELISA‐positive samples were positive with either Advate or NovoEight. Additionally, 10.7%–14.3% were xFLI‐positive and ELISA‐negative. All but one CBA‐positive sample was above 3SD with xFLI; one was between 2 and 3SD. 29.1% were xFLI‐positive and CBA negative. The overall concordance between xFLI and ELISA was 82.1% and xFLI and CBA 77.9%. Conclusion: The anti‐FVIII antibody xFLI method is adaptable to clinical practice and more sensitive and reproducible than ELISA and CBA. Actual NNA titers are determined to both full‐length and B‐domain deleted FVIII. The xFLI is thus valuable for confirmation of all anti‐FVIII antibodies. [ABSTRACT FROM AUTHOR]

Introduction: Monitoring replacement therapy with standard and extended half‐life (EHL) products is challenging, since one‐stage assay (OSA) and chromogenic substrate assay (CSA) results may differ significantly. Recent recommendations include local validation of each new product with recovery within 20–30%, depending on activity level. Aim: To validate factor VIII (FVIII) activity for monitoring products in clinical use on Atellica Coag and to correlate it with thrombin generation. Methods: Plasma samples spiked with Advate®, Elocta®, Adynovi®, Nuwiq®, NovoEight® and Afstyla® (0.05, 0.20, 0.50 and 0.80 IU/ml) were analysed using Atellica Coag 360 with CSA‐1 (Coatest SP) and CSA‐2 (FVIII chromogenic), and OSA (Actin FS). Thrombin generation was performed using two thrombin generation assays (TGA‐1 (Thrombinoscope) and TGA‐2 (Technothrombin). Results: All products at levels above 0.05 IU/ml, except Adynovi, showed acceptable recovery using CSA‐1, whereas measurements using CSA‐2 gave more results outside the target level. All products, except Afstyla, showed acceptable recovery using OSA. Correlation between CSA‐1 and OSA was excellent (r2=1.0) with biases of 6–3​2%, depending on FVIII product. A clear dose‐response was seen for all thrombin generation parameters and products using both methods, except at low levels for lag time using TGA‐1. With CSA‐1 as an independent variable, the correlations to thrombin peak (measured with TGA‐2) were good (r2 =.8–.9). Conclusion: Our data revealed good correlation and acceptable bias between CSA and OSA using our sets of reagents, methods and analyser in spiked samples. Thrombin generation gave good correlation to CSA‐1 factor activity and is a possible complement to factor activity assays. [ABSTRACT FROM AUTHOR]