Development of a rapid and fully automated factor VIII inhibitor assay, insensitive to emicizumab, and a lowest level of quantification of 0.2 BU/mL.

BACKGROUND: Factor (F)VIII inhibitors are measured using labor- and resource-expensive Nijmegen or Bethesda assays, which lack sensitivity for low-titer inhibitors and show high variations in quality surveys, mainly because of manual assay procedures. OBJECTIVES: The goal of this study was the development of a fast and fully automated FVIII inhibitor assay by using recombinant (r)FVIII as substrate and dedicated equipment for execution of the test. METHODS: A new rapid, fully automated, FVIII inhibitor assay is presented, the core of which is use of full-length recombinant FVIII (rFVIII; Kovaltry, Bayer) as inhibitor substrate instead of plasma FVIII, resulting in rapid binding of inhibitors to rFVIII due to absence of von Willebrand factor. Dramatic shortening of incubation time facilitated full automation on an analyzer capable of 3 subsequent sample dilution steps and 3 reagent additions. Equal volume mixtures of sample and rFVIII (1.0 U/mL) were incubated for 10 minutes at 37 °C, whereafter remaining FVIII activity was analyzed with a kinetic chromogenic assay, allowing inhibitor activity calculation without preceding FVIII activity calibration, using a Ceveron s100 analyzer (Technoclone). RESULTS: Mean titer in 60 nonhemophiliacs was 0.0 BU/mL (SD, 0.1), yielding a limit of blank of 0.1 BU/mL and lower limit of quantification of 0.2 BU/mL. Analyses were performed with the new method and a Nijmegen assay in 28 inhibitor-positive clinical samples, 14 containing emicizumab and 14 without. Correlation coefficient in emicizumab-free type I inhibitor samples was 1.0. Emicizumab dependency of the method was excluded in spiking experiments with inhibitor-positive samples. Reproducibility was tested by analyzing 7 samples in 3 laboratories for 5 days, twice daily; coefficients of variation of all samples were <15%. CONCLUSION: We present development data of a sensitive and specific rapid, automated FVIII inhibitor assay generating results within 20 minutes that is less resource-intensive than standard assays with potential to improve assay variability.

Prediction of inhibitor development in previously untreated and minimally treated children with severe and moderately severe hemophilia A using a machine-learning network.

BACKGROUND: Prediction of inhibitor development in patients with hemophilia A (HA) remains a challenge. OBJECTIVES: To construct a predictive model for inhibitor development in HA using a network of clinical variables and biomarkers based on the individual similarity network. METHODS: Previously untreated and minimally treated children with severe/moderately severe HA, participants of the HEMFIL Cohort Study, were followed up until reaching 75 exposure days (EDs) without inhibitor (INH-) or upon inhibitor development (INH+). Clinical data and biological samples were collected before the start of factor (F)VIII replacement (T0). A predictive model (HemfilNET) was built to compare the networks and potential global topological differences between INH- and INH+ at T0, considering the network robustness. For validation, the "leave-one-out" cross-validation technique was employed. Accuracy, precision, recall, and F1-score were used as evaluation metrics for the machine-learning model. RESULTS: We included 95 children with HA (CHA), of whom 31 (33%) developed inhibitors. The algorithm, featuring 37 variables, identified distinct patterns of networks at T0 for INH+ and INH-. The accuracy of the model was 74.2% for CHA INH+ and 98.4% for INH-. By focusing the analysis on CHA with high-risk F8 mutations for inhibitor development, the accuracy in identifying CHA INH+ increased to 82.1%. CONCLUSION: Our machine-learning algorithm demonstrated an overall accuracy of 90.5% for predicting inhibitor development in CHA, which further improved when restricting the analysis to CHA with a high-risk F8 genotype. However, our model requires validation in other cohorts. Yet, missing data for some variables hindered more precise predictions.

Current status of haemophilia inhibitor management in mainland China: a haemophilia treatment centres survey on treatment preferences and real-world clinical practices.

To investigate the current experience and expertise for haemophilia inhibitor patient management in haemophilia treatment centres (HTCs) in mainland China. Questionnaires were distributed to 'tertiary tier A' hospital HTCs across China to collect information on treatment preferences for bleeding control, prophylaxis and inhibitor eradication, as well as their regimens in real-world clinical practice. Of 40 questionnaires distributed, 39 were returned. In all, 38 were analysable for treatment preferences and 34 for actual clinical practice. For haemostatic treatment, 76·3% (29/38) HTCs preferred activated recombinant human Factor VII (rFVIIa). In clinical practice, the most widely used by-pass agent was prothrombin complex concentrate (26 HTCs). Although 65·8% (25/38) of HTCs believed prophylaxis treatment was necessary, it was prescribed in only 12. Similarly, 65·8% (25/38) of HTCs believed immune tolerance induction (ITI) therapy was necessary but only 14·8% (92/622) of patients in 19 HTCs received low-dose ITI treatment. HTCs in relatively economically developed cities (with higher-than-average per-capita gross domestic product) had better access to haemostatic treatment, coagulation testing and were more likely to provide prophylaxis and ITI in practice. The present survey showed there were gaps in haemophilia inhibitor care between the HTC physicians' preferences and their actual clinical practice. More specific care guidelines, education and clinical decision support tools are needed to guide clinical practice.

COVID-19 Convalescent Plasma Is More than Neutralizing Antibodies: A Narrative Review of Potential Beneficial and Detrimental Co-Factors.

COVID-19 convalescent plasma (CCP) is currently under investigation for both treatment and post-exposure prophylaxis. The active component of CCP mediating improved outcome is commonly reported as specific antibodies, particularly neutralizing antibodies, with clinical efficacy characterized according to the level or antibody affinity. In this review, we highlight the potential role of additional factors in CCP that can be either beneficial (e.g., AT-III, alpha-1 AT, ACE2+ extracellular vesicles) or detrimental (e.g., anti-ADAMTS13, anti-MDA5 or anti-interferon autoantibodies, pro-coagulant extracellular vesicles). Variations in these factors in CCP may contribute to varied outcomes in patients with COVID-19 and undergoing CCP therapy. We advise careful, retrospective investigation of such co-factors in randomized clinical trials that use fresh frozen plasma in control arms. Nevertheless, it might be difficult to establish a causal link between these components and outcome, given that CCP is generally safe and neutralizing antibody effects may predominate.

Hemostatic Efficacy and Anti-FXa (Factor Xa) Reversal With Andexanet Alfa in Intracranial Hemorrhage: ANNEXA-4 Substudy.

Background and Purpose: Andexanet alfa is a recombinant modified human FXa (factor Xa) developed to reverse FXa inhibition from anticoagulants. Hemostatic efficacy and reversal of anti-FXa activity with andexanet were assessed in patients from the ANNEXA-4 study (Andexanet Alfa, a Novel Antidote to the Anticoagulation Effects of FXa Inhibitors) with intracranial hemorrhage (ICrH). Methods: ANNEXA-4 was a single-arm study evaluating andexanet in patients presenting with major bleeding ≤18 hours after taking an FXa inhibitor. Patients received a bolus plus 2-hour infusion of andexanet. Brain imaging in patients with ICrH was performed at baseline and at 1 and 12 hours postandexanet infusion. Coprimary efficacy outcomes were change in anti-FXa activity and hemostatic efficacy at 12 hours (excellent/good efficacy defined as ≤35% increase in hemorrhage volume/thickness). Safety outcomes included occurrence of thrombotic events and death at 30 days. Results: A total of 227 patients with ICrH were included in the safety population (51.5% male; mean age 79.3 years) and 171 in the efficacy population (99 spontaneous and 72 traumatic bleeds). In efficacy evaluable patients, excellent/good hemostasis 12 hours postandexanet occurred in 77 out of 98 (78.6%) and in 58 out of 70 (82.9%) patients with spontaneous and traumatic bleeding, respectively. In the subanalysis by FXa inhibitor treatment group in the efficacy population, median of percent change in anti-FXa from baseline to nadir showed a decrease of 93.8% for apixaban-treated patients (n=99) and by 92.6% for rivaroxaban-treated patients (n=59). Within 30 days, death occurred in 34 out of 227 (15.0%) patients and thrombotic events occurred in 21 out of 227 (9.3%) patients (safety population). Conclusions: Andexanet reduced anti-FXa activity in FXa inhibitor-treated patients with ICrH, with a high rate of hemostatic efficacy. Andexanet may substantially benefit patients with ICrH, the most serious complication of anticoagulation. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02329327.

Characterisation and application of recombinant FVIII-neutralising antibodies from haemophilia A inhibitor patients.

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products.

First observation of inhibitor development against efmoroctocog alfa in France.

In patients with severe haemophilia receiving clotting factor concentrates, the risk of immunisation against their usual treatment is still patent and feared. New haemophilia drug treatments with an extended half-life have become available over the past few years. The risk of inhibitor development to these new treatments is unclear. We report the case of a 51-year-old man with severe haemophilia A, who was previously treated with no history of inhibitor development. Soon after a switch in his treatment to efmoroctocog alfa he developed an inhibitor against this recombinant Fc fusion extended half-life FVIII (rFc-FVIII) product. The patient was on an on-demand treatment regimen and was treated for mucosal bleeding. The inhibitor was characterised as type I, with classical epitope mapping. The spontaneous evolution of this inhibitor was favourable, but an anamnestic response led to a switch in his treatment to emicizumab.

Clot waveform analysis for perioperative hemostatic monitoring of arthroscopic synovectomy in a pediatric patient with hemophilia A and inhibitor receiving emicizumab prophylaxis.

Emicizumab reduces bleeding in patients with hemophilia A and inhibitors (PwHA-I). Coagulation potential during the perioperative period in emicizumab-treated PwHA-I undergoing surgery remains to be evaluated. We describe a 14-year-old boy with HA-I receiving emicizumab prophylaxis who experienced arthroscopic synovectomy. He was treated with a bolus infusion of recombinant factor VIIa (rFVIIa; 80 µg/kg) immediately before surgery, and treatment continued at the same dose every 3 h on day 1, every 4 h on day 2, and every 6 h on day 3. Treatment with rFVIIa was discontinued on day 4. No perioperative bleeding or thrombotic events were observed. Coagulation potential throughout the perioperative period was retrospectively assessed with an easy-to-use clot waveform analysis (CWA). Measurements from CWA returned to within or near the normal range, suggesting successful hemostatic management. Coagulation potentials assessed by CWA showed a significant correspondence with those from a thrombin generation assay (TGA) that is already in use. CWA and TGA could both provide useful data for assessing coagulation potential in the perioperative hemostatic management of emicizumab-treated PwHA-I.

Treatment of acquired hemophilia A, a balancing act: results from a 27-year Dutch cohort study.

Acquired hemophilia A (AHA) is a severe auto-immune bleeding disorder. Treatment of AHA is burdensome and optimal management is still unresolved. Therefore a retrospective nationwide multi-center cohort study (1992-2018) was performed to evaluate clinical presentation and treatment efficacy and safety of AHA in the Netherlands. Multivariate logistic and Cox regression analysis was used to study independent associations between patient characteristics and clinical outcomes. A total of 143 patients (median age 73 years; 52.4% male) were included with a median follow-up of 16.8 months (IQR 3.6-41.5 months). First-line immunosuppressive treatment was mostly steroid monotherapy (67.6%), steroids/cyclophosphamide (11.9%) and steroids/rituximab (11.9%), with success rates of 35.2%, 80.0% and 66.7% respectively, P < .05. Eventually 75% of patients achieved complete remission (CR). A high anti-FVIII antibody titer, severe bleeding and steroid monotherapy were associated with lower CR rates. Infections, the most important adverse event, occurred significantly more often with steroid combination therapy compared to steroids alone (38.7% vs 10.6%; P = .001). Overall mortality was 38.2%, mostly due to infections (19.2%) compared to 7.7% fatal bleeds. Advanced age, underlying malignancy and ICU admission were predictors for mortality. This study showed that AHA is characterized by significant disease-related and treatment-related morbidity and mortality. A high anti-FVIII titer, severe bleeding and steroid monotherapy were associated with a lower CR rate. The efficacy of steroid combination therapies however, was overshadowed by higher infection rates and infections represented the most important cause of death. The challenging and delicate balance between treatment effectivity and safety requires ongoing monitoring of AHA and further identification of prognostic markers.

Prophylaxis Using a Mixture of Plasma-Derived Activated Factor VII and Factor X (pdFVIIa/FX) in a Patient with Hemophilia B Complicated by Inhibitors and Allergy to Factor IX Concentrates: A Case Report.

Treating patients with hemophilia and inhibitors is often problematic. The presence of inhibitors negatively impacts the effectiveness of treatment to achieve hemostasis especially in patients with hemophilia B, owing mainly to allergic reactions to factor IX (FIX) concentrates and the low success rate of immune tolerance therapy. A 9-month-old boy had intracranial hemorrhage and was diagnosed with hemophilia B. After replacement therapy, he developed inhibitors and an allergic reaction to FIX. Prophylactic therapy was initiated with recombinant activated factor VII (rFVIIa) and later switched to pdFVIIa/factor X (FX; 120 µg/kg as the FVII dose, every other day) because of a recurrence of intracranial hemorrhage. Since then, he remained well without life-threatening bleeding for more than 2 years. Our case suggests that pdFVIIa/FX may be useful for prophylactic therapy in hemophilia B complicated by inhibitors and allergic reaction to FIX concentrates.

Congenital Factor XIII Deficiency With the Presence of Inhibitor: A Case Study.

Coagulation factor XIII (FXIII) is a fibrin-stabilizing factor with additional roles in wound healing and interactions between the decidua and fetus. Congenital FXIII deficiency is rare bleeding disorder. Inhibitor development against FXIII in inherited FXIII deficency is also uncommon, but may cause severe, life-threatening bleeding. FXIII is the last step in the coagulation cascade with normal coagulation paramaters (PT, aPTT), the detection of inhibitor to FXIII is quite difficult. The treatment of inhibitor-positive congenital FXIII deficiency is challenging due to the lack of a role of by-pass agents such as FVII. The best known ways of treatment in these cases are the use of high-dose FXIII concentrates and immunosuppression. Herein, we report the management of postoperative bleeding diathesis in a patient with FXIII deficiency who developed inhibitors, and to follow the clinical course of the disease with FXIII concentrate and immunosuppression.

Profile of natural anticoagulant, coagulant factor and anti-phospholipid antibody in critically ill COVID-19 patients.

The outbreak of novel coronavirus disease 2019 (COVID-19) has now become a global pandemic. Coagulopathy has been reported widely in critically ill COVID-19 patients and was related to high mortality. However, the comprehensive coagulation profiles have not been examined and the underlying mechanism of the coagulopathy in COVID-19 patients is unclear. To study the coagulation profiles of routine hemostasis tests, natural anticoagulants, coagulant factors and antiphospholipid antibodies in critically ill COVID-19 patients. This single-center and cross-section study included 19 patients with COVID-19, who were admitted to intensive care unit (ICU) at Tongji hospital in Wuhan, China, from Feb 23 to Mar 3, 2020. Demographic data, laboratory parameters, treatments and clinical outcomes of the patients were collected and analyzed. The final date of follow-up was Mar 31, 2020. In this study, 12 thrombotic events occurred in 9 patients, including 4 cerebral infarctions, 7 acro-ischemia and 1 internal jugular vein thrombosis. The common abnormalities of routine coagulation tests included evelated D-Dimer level (100%), prolonged prothrombin time (73.7%) and hyperfibrinogenemia (73.7%). The median activities of natural anticoagulants including protein C, protein S and antithrombin were all below the normal range. Factor VIII activities were significantly above normal range (median value 307%, IQR 198-441) in all patients. Factor V and factor VII activities were significantly lower in near-terminal stage patients. Anti-phospholipid antibodies were present in 10 patients. Strikingly, 4 cerebral infarction events were in patients had anti-phospholipid antibodies of multiple isotypes. Sustained hypercoagulable status and thrombotic events were common in critically ill patients with COVID-19. The low activities of natural anticoagulants, elevated factor VIII level and the presence of antiphospholipid antibodies, together, may contribute to the etiopathology of coagulopathy in COVID-19 patients.

Bleeding and response to hemostatic therapy in acquired hemophilia A: results from the GTH-AH 01/2010 study.

Acquired hemophilia A (AHA) is due to autoantibodies against coagulation factor VIII (FVIII) and most often presents with unexpected bleeding. In contrast to congenital hemophilia, the patient's residual FVIII activity does not seem to correlate with the risk of bleeding as suggested from previous studies. Risk factors for bleeding have not been described. We used data from the prospective GTH-AH 01/2010 study to assess the risk of bleeding and the efficacy of hemostatic therapy. FVIII activity was measured at baseline and weekly thereafter. Bleeding events were assessed by treating physicians. A total of 289 bleeds were recorded in 102 patients. There were 141 new bleeds observed starting after day 1 in 59% of the patients, with a mean rate of 0.13 bleed per patient-week in weeks 1 to 12, or 0.27 bleed per patient-week before achieving partial remission. Weekly measured FVIII activity was significantly associated with the bleeding rate, but only achieving FVIII activity ≥50% abolished the risk of bleeding. A good World Health Organization performance status assessed at baseline (score 0 vs higher) was associated with a lower bleeding rate. Hemostatic treatment was reportedly effective in 96% of bleeds. Thus, the risk of new bleeds after a first diagnosis of AHA remains high until partial remission is achieved, and weekly measured FVIII activity may aid in assessing the individual risk of bleeding. These results will help to define future strategies for prophylaxis of bleeding in AHA.

Advances in managing rare acquired bleeding disorders.

INTRODUCTION: Rare acquired bleeding disorders include a wide spectrum of coagulopathies characterized by spontaneous or post-trauma and post-surgery hemorrhages in patients without a previous personal or family history of bleeding. AREAS COVERED: This review, based on a Medline/PubMed search during the last 20 years, will focus mainly on rare acquired bleeding disorders caused by autoantibodies against coagulation factors, including autoantibodies against factor VIII (acquired hemophilia A), von Willebrand factor (acquired von Willebrand syndrome) and other coagulation factors (factors V, X, XI, and XIII). The pathogenic, laboratory, and clinical features of these rare hemorrhagic conditions will be discussed, with particular attention to their management. EXPERT OPINION: The treatment of rare acquired bleeding disorders includes the control of bleeding and the elimination of the autoantibody and of the underlying disease, when present. As the bleeding clinical phenotype is often severe, the management of affected patients is particularly challenging. Thus, while an early diagnosis of the acquired coagulopathy is essential to start the most appropriate treatment and to improve patients' outcomes, the support of specialized centers is equally important to provide a correct management of such complicated cases.

Evaluation of activated partial thromboplastin time coagulation waveform analysis for identification of patients with acquired factor VIII inhibitors.

INTRODUCTION: Activated partial thromboplastin time (PTT) coagulation waveforms produced by optical detection system coagulation analyzers provide additional potentially useful and routinely underutilized information for the evaluation of a patient's coagulation system. We aimed to identify features of PTT coagulation waveforms, available for all PTT assays performed in our hospital laboratories, that may prove useful in directing early investigations in patients with unexplained prolonged PTT. METHODS: We retrospectively reviewed 211 PTT coagulation waveforms from patient testing and categorized them based on the underlying hemostatic abnormality: normal, therapeutic anticoagulation, lupus anticoagulant, congenital factor deficiency, or acquired factor VIII inhibitor. We compared quantitative waveform parameters and the frequency of qualitatively abnormal double-peaked first derivative waveform curves between these groups. RESULTS: Partial thromboplastin time and derivative curve maxima and minima differed significantly between acquired factor VIII inhibitors and other diagnostic categories, and the second derivative curve minimum demonstrated the highest area under the receiver operator characteristic curve for identification of acquired factor VIII inhibitors (0.860; maximum accuracy: 79.5% for 2Dmin> -39.3 mAbs/s2 [sensitivity 90.5%; specificity 77.2%]). The presence of an abnormal double-peaked first derivative curve had a sensitivity of 83.3% and specificity of 81.6% for identification of acquired factor VIII inhibitors in cases with PTT >50 seconds. CONCLUSION: Partial thromboplastin time coagulation waveform analysis can aid in identification of patients with acquired factor VIII inhibitors and may be of clinical utility in directing early laboratory investigations to identify patients at risk of severe bleeding without prompt intervention.