Clotting Factor Replacement Therapy Research Articles

The use of Bacillus subtilis as a cost-effective expression system for production of Cholera Toxin B fused factor VIII epitope regions applicable for inducing oral immune tolerance.

Coagulation factor replacement therapy for the X-linked bleeding disorder Haemophilia, characterized by a deficiency of coagulation protein factor VIII (FVIII), is severely complicated by antibody (inhibitors) formation. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with a tremendous increase in morbidity as well as treatment costs. The ultimate goal of inhibitor control is antibody elimination. Immune tolerance induction (ITI) is the only clinically established approach for developing antigen-specific tolerance to FVIII. This work aims to establish a novel cost-effective strategy to produce FVIII molecules in fusion with cholera toxin B (CTB) subunit at the N terminus using the Bacillus subtilis expression system for oral tolerance, as the current clinical immune tolerance protocols are expensive. Regions of B-Domain Deleted (BDD)-FVIII that have potential epitopes were identified by employing Bepipred linear epitope prediction; 2 or more epitopes in each domain were combined and cDNA encoding these regions were fused with CTB and cloned in the Bacillus subtilis expression vector pHT43 and expression analysis was carried out. The expressed CTB-fused FVIII epitope domains showed strong binding affinity towards the CTB-receptor GM1 ganglioside. To conclude, Bacillus subtilis expressing FVIII molecules might be a promising candidate for exploring for the induction of oral immune tolerance.

Analysis of Perioperative Factor Replacement Therapy for Hemophilia

Background Hemophilia is a hereditary bleeding disorder, especially in severe hemophilia, that can lead to spontaneous bleeding. For hemophilia patients with inhibitors, even minor acute bleeding events or surgical procedures can have catastrophic consequences. Some hemophiliacs are forced to make surgical choices. There are many risks in perioperative period when hemophiliacs undergo major surgery. Seeking effective and safe perioperative clotting factor replacement therapy or bypass therapy is the basis of successful surgery. Aims To seek effective and safe perioperative coagulation factor replacement therapy or bypass therapy. Methods HA patients were treated with FVIII preparation during perioperative period. To calculate dosage, multiply the patient ' s weight in kilograms by the FVIII level in IU/dL desired, then multiply by 0.5. Example: 50 kg × 40 (IU/dL level desired) × 0.5 = 1000 IU of FVIII. HB patients were treated with PCC or FIX during perioperative period. To calculate dosage, multiply the patient ' s weight in kilograms by the FIX level in IU/dL desired. Example: 50 kg body weight × 40 (IU/dL level desired) = 2000 IU of plasma-derived FIX. For hemophilia patients with low titer inhibitors, estimate the amount of FVIII required as a neutralizing inhibitor loading dose as recommended by WFH. The following formula is used to estimate the amount of FVIII needed as a loading dose to neutralize the inhibitor: [body weight (kg) × 80 × [(1 − hematocrit) × antibody titer (BU)]. An additional 50 IU/kg above the calculated loading dose is added to achieve a measurable FVIII activity. Patients with hemophilia combined with high titer inhibitors were treated with rFVIIa combined with PCC SCBT regimen. Sequential use refers to alternating rFVIIa and aPCC every 3 hours. Results 69 patients with indications for surgery and/or desire for surgery who underwent different types of surgery with clotting factor replacement therapy or bypass therapy were evaluated (Table 1). There were 37 cases of severe hemophilia and 32 cases of non-severe hemophilia. There were 63 cases of HA, including 50 cases of negative inhibitor and 13 cases of positive inhibitor. After meeting the inclusion and exclusion criteria, 69 patients were evaluated for different types of surgery under clotting factor replacement therapy or bypass therapy during perioperative period. 59 cases underwent major operations, including: 49 cases underwent joint replacement, among which 34 cases underwent single joint replacement and 6 cases underwent double joint replacement and so on. 10 cases underwent minor operations. There were 14 cases of hemophilia with inhibitors, of which 13 were major operations with 175.38 (85.00-270.00) minutes of operation and 2 were minor operations with 45.00 minute of operation. In 12 patients with severe hemophilia and high titer inhibitors, the perioperative consumption of factor FVII was as follows: 447.62 (331.04-738.48)ug/kg on the day of surgery, and the total consumption was 2484.21 (1499.40-4500.00)ug/kg. The perioperative consumption of factor PCC was as follows: 126.10 (0-206.45)ug/kg on the day of surgery, and the total consumption was 1332.84 (529.20-1970.00)ug/kg. The mean preoperative FVIII:C level of the HA patients was 1.19 (0.00~6.00)%, the mean preoperative FIX:C level was 0.022 (0.01~0.07)% of the HB patients.The average intraoperative blood loss of the 69 patients was 537.98 (0-3000) ml. 20 patients received intraoperative transfusion of blood products (red blood cells or fresh frozen plasma) and 49 patients did not receive intraoperative transfusion of blood products. In 55 hemophiliacs without inhibitor, no inhibitor was found after surgery. Conclusion(s) It is safe and effective to adopt different schemes for perioperative alternative therapy for different hemophilia types.

Assessment of Transduction of CD34+ Human Hematopoietic Stem Cells from Patients with Severe Hemophilia-Α with Lentiviral Vector Carrying a High Expression FVIII Transgene (CD68-ET3-LV)

Background: Alternative strategies are needed for the large number of people with hemophilia who are ineligible for AAV based gene therapy due to age or high levels of anti-AAV neutralizing antibodies. High inter-individual variability of expression and ill sustained factor levels are also challenges with AAV based gene therapy for hemophilia A, in particular. We have developed a third-generation lentiviral vector mediated hematopoietic stem cell-based gene therapy for hemophilia A. (Doering et al Human Gene Therapy 2018; 29: 1183-1201) This vector (CD68-ET3-LV) has a high expression FVIII transgene with a CD68 promoter targeting expression in monocytic cells predominantly. We report here successful transduction of severe hemophilia A patient derived human hematopoietic stem cells with this vector. Methods: Mobilized peripheral blood stem cells were collected by apheresis from three patients with severe hemophilia A without inhibitors who also received prophylactic clotting factor replacement therapy during this period. CD34+ hematopoietic stem cells (HSCs) were enriched on the CliniMACS Plus® system (Miltenyi Biotec, Bergish Gladbach, Germany) using the CliniMACS CD34® reagent system. Purified HSC were then transduced with this CD68-ET3-LV as follows - HSCs cultured on retronectin coated surfaces in xenofree media with cytokines were exposed to clinical grade CD68-ET3-LV vector in two ways - a double transduction protocol without any enhancer and a single transduction with an enhancer. After completion of this step, the product was assessed for viability and vector copy number (VCN) by trypan blue labelling and Q-PCR performed on genomic DNA from CFU cells, respectively. Transduced HSCs were also assessed for their engraftment potential. CD68-ET3-LV vector transduced HSC (1x10 6) were transplanted into NBSGW mice via tail vein injection. Engraftment was assessed at 16 weeks after transplantation. This protocol was approved by the Institutional Review Board of the Christian Medical College, Vellore, India. Results: G-CSF (10 ug/kg/day) based peripheral blood stem cell mobilization was well tolerated by all participants. The apheresis procedures which were done with plasma FVIII levels in the normal range after prophylactic replacement therapy were unremarkable. The total collection of mobilized CD34+ cells from the three donors was 268±80.5x10 6 CD34+ cells. An aliquot of 2x10 6 CD34+ cells/ml was used in the transduction experiments. The data on viability and vector copy number post transduction shown in the tables 1 and 2 confirm that viability was not affected by the manipulation of these HSCs in both double and single transduction methods. The vector copy number (VCN) in the genomic DNA obtained from CFU cells collected from CD34+ HSCs cultured in MethoCult H4434™ (Stem Cell Technologies™, Vancouver, Canada) varied from 0.62 to 1.21 in double transduction (n=3) and nearly doubled in the single transduction method with an enhancer to VCNs of 1.5 and 2.4 in two samples tested. Transplantation studies in NBSGW mice showed successful engraftment of human CD34+ HSCs with a mean engraftment of 80.99 ± 4.2% of CD45+ multilineage hematopoietic cells in the bone marrow 16 weeks after transplantation. Conclusion: To the best of our knowledge, this is the first report of transduction of mobilized peripheral blood CD34+ HSCs from patients with severe hemophilia A using a clinical grade lentiviral vector with a high expression FVIII transgene. These data establish feasibility and safety of the procedure. The transduction protocol showed good efficiency with very high viability, significant VCN and good engraftment of these gene modified human HSCs in a mouse model. These protocols are currently being evaluated in a first in human phase 1 clinical trial of gene therapy for severe hemophilia A without inhibitors.

Factor First: An Assessment of Emergency Department Care of Patients with Hemophilia

Background: With the establishment of hemophilia treatment centres (HTCs) and home infusion programs for factor replacement therapy, emergency department use among patients with hemophilia occurs less frequently, with patients often presenting for significant bleeding events only. As a result, the management of patients with hemophilia can represent a challenge to emergency department (ED) physicians and staff. Both delays in diagnosis of a bleeding event, and timely administration of hemostatic therapy have been associated with patient morbidity and mortality. The World Federation of Hemophilia, in addition to other organizations, emphasize the importance of timely receipt of clotting factor replacement therapy, providing treatment at the time a bleed is suspected. If a patient with hemophilia is registered with a HTC, they often carry a wallet card, or have a formalized treatment plan, outlining their diagnosis and emergency treatment of bleeding recommendations to ensure timely access to hemostatic therapy. The aim of this study was to describe ED management of patients with hemophilia at a single Canadian HTC, with a focus on use of individualized emergency bleeding management protocols, and time to receipt of hemostatic therapy within the ED. Methods: Adult patients with hemophilia A or B (FVIII or FIX ≤40%), of any severity, followed at the Southern Alberta Rare Blood and Bleeding Disorders Comprehensive Care Program, were identified for inclusion. ED health utilization and ED encounter management data for each of the four hospital sites in Calgary, AB was obtained from the electronic health record for the cohort, through to 2022. Results: A total of 191 patients with hemophilia A and B were identified for study inclusion. 153 patients with hemophilia A (mild 64.7%; moderate 5.9%; severe 28.8%) and 38 patients with hemophilia B (mild 44.7%; moderate 36.8%; severe 18.4%). Males comprised 77.5% of the patient cohort. A total of 393 ED visits occurred over the study period. Less than a third of ED visits were associated with receipt of hemostatic therapy (either desmopressin or clotting factor concentrate). The median time from triage to ordering of hemostatic therapy was 2.9 hrs (IQR 2.3-3.2), and median time from triage to administration of hemostatic therapy was 4.2 hrs (IQR 3.1-4.2). For ED visits due to suspected bleeds, the proportion of patients who received either coagulation factor replacement or desmopressin within two hours of ED triage is depicted in Figure 1. Time to receipt of hemostatic therapy did not significantly differ whether desmopressin or clotting factor concentrate was administered. The number of ED visits in which the individualized emergency bleeding management protocol was accessed is depicted in Figure 2. Among those accessed in 2022, the proportion of protocols that were updated within the last 365 days was 86.8%. Less than 20% of ED visits were associated with a hematology consultation. Conclusions: Increasing utilization of individualized emergency bleeding management protocols for patients with hemophilia was observed; however, significant delays in timely receipt of hemostatic therapy remains, with infrequent involvement of hematology in consultation. Time to receipt of potentially life-saving hemostatic therapy for our cohort falls outside of hemophilia care standards. This data provides a better understanding of the experience of patients with hemophilia in the ED and provides an opportunity to define further quality improvement interventions to ensure quality care metrics in this population are met.

Acute coronary syndrome management in hemophiliacs: How to maintain balance?: A review.

To analyze existing literature and understand how to balance the minimization of bleeding risk and ensuring adequate anticoagulation during coronary intervention treatment and long-term postoperative anticoagulation in hemophilia patients during the perioperative period, in order to provide guidance for healthcare professionals in developing effective treatment plans. This narrative review will analyze existing studies, case reports, and clinical guidelines to determine the most effective strategies for managing acute coronary syndrome in hemophilia patients. When evaluating the literature, factors such as patient age, medical history, and severity of the condition will be considered. The current management guidelines for acute coronary syndrome in hemophilia patients are not based on systematic evaluation and mainly rely on expert opinions. This article provides a comprehensive analysis of existing literature and recommends coagulation factor replacement therapy before anticoagulation and intervention treatment, as well as personalized anticoagulation treatment during the postoperative period for better management of hemophilia patients with acute coronary syndrome. These recommendations can help healthcare professionals develop more effective treatment plans for hemophilia patients.

Diagnosis and treatment of autoimmune acquired coagulation factor deficiency

Autoimmune coagulation factor deficiency (AiCFD) is an acquired bleeding disorder caused by immunoglobulins (autoantibodies) that target a single coagulation factor. Most of these autoantibodies are polyclones and primarily neutralizing antibodies (inhibitors) that inhibit the function of coagulation factors; however, non-neutralizing autoantibodies that enhance clearance are also present. AiCFD has been reported in nearly all coagulation factors and von Willebrand factor, and its representative disease is acquired hemophilia A, which is caused by autoantibodies against coagulation factor VIII. The treatment for AiCFD consists of hemostatic therapy according to the bleeding symptoms and immunosuppressive therapy to eradicate autoantibodies. Hemostatic treatment varies depending on the deficient coagulation factor, and coagulation factor replacement therapy, platelet or fresh frozen plasma transfusions, and bypassing agents are provided. Although AiCFD is a rare disease, raising awareness of this disease is necessary because general physicians may also encounter it.

Effectiveness of Prophylactic Coagulation Factor Replacement Therapy in Patients with Severe Hemophilia A in Taiwan - A Population-Based Study.

Taiwan launched reimbursement of prophylactic coagulation factor replacement therapy (CFRT) for patients with severe hemophilia type A (severe PWHA) in 2014. However, since then, the effectiveness of prophylactic CFRT in real-world practice has not been evaluated thoroughly. This study aimed to evaluate the effectiveness of prophylactic CFRT in severe PWHA cases on the outcome of bleeding risks. We included male, severe PWHA cases from a nationwide, population-based database in Taiwan. Given that the database lacked details of the dosing regimen for prophylactic CFRT, we applied group-based trajectory modeling using the proportion of days covered (PDC) by CFRT from 2014 to 2015 in order to classify patients. A high PDC level corresponded to a greater proportion of time under CFRT, thus implying that the patient was probably receiving prophylactic therapy. We followed up patients from January 01, 2016 until occurrence of any bleeding events, death or December 31st 2017. We identified a total of 420 severe PWHA and classified them into high- (n = 88), medium- (n = 181) and low- (n = 151) PDC groups. The mean (±SD) PDC values of the three groups were 0.78 (±0.1), 0.40 (±0.1) and 0.12 (±0.1), respectively. Using Cox regression models with propensity score adjustment, we found patients with medium- (hazard ratio: 0.69; 95% CI: 0.56-0.89) or high-PDC (0.45; 0.36-0.68) under CFRT had reduced risks of any bleeding, compared to the low PDC group. The findings demonstrated the effectiveness of prophylactic CFRT in the prevention of bleeding events in real-life severe PWHA.

Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology

BackgroundHemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X chromosome. Hemophilia B is currently incurable and the standard treatment is coagulation factor replacement therapy. Although gene therapy has the potential to cure hemophilia, significant barriers are still needed to be overcome, e.g., off-target effects and immunoreactivity, so new approaches must be explored. Nonsense mutations account for 8% of all the hemophilia B mutation types and can result in the development of coagulation factor inhibitors. In this study, CRISPR/Cas9 technology was used to construct a mouse embryonic stem cell model with a hemophilia B nonsense mutation (F9 c.223C > T) in humans to investigate the pathogenesis and treatment of nonsense mutations in hemophilia B.MethodsFirst, a donor plasmid with a mutation (F9 c.223 C > T) and sgRNAs were constructed. Second, both the donor plasmid and the px330-sgRNA were electroporated into mouse embryonic stem cell, and the mutant cells were then screened using puromycin and red fluorescence. Third, the mutant cell lines were tested for pluripotency and the ability to differentiate into three layers. Finally, the effect of mutation on gene function was studied in the differentiation system.ResultsThe mutant vector and effective sgRNA were constructed, and the mutant cell line was screened. This mutant cell line exhibited pluripotency and the ability to differentiate into three layers. This point mutation affects F9 expression at both the RNA and protein levels in the differentiation system.ConclusionsThe mutant cell line obtained in the current study had a single-base mutation rather than a base deletion or insertion in the exon, which is more similar to clinical cases. In addition, the mutant has the characteristics of mouse embryonic stem cells, and this point mutation affects F9 gene transcription and translation, which can be used as a disease model for studying the pathogenesis and treatment of hemophilia at the stem cell level.

EE476 Cost-Effectiveness of Emicizumab Prophylaxis in Patients with Hemophilia a without Inhibitors

Emicizumab is a novel monoclonal antibody approved for the prevention and treatment of bleeds in patients with hemophilia A with or without inhibitors. The development of inhibitors, which are neutralizing antibodies to clotting factor replacement therapy, is a major treatment-related complication in hemophilia management. In patients with inhibitors, bleed prophylaxis using emicizumab has been shown to be cost-effective compared to prophylaxis using bypassing agents – with supporting evidence for its reimbursement in this population in Canada.

Emicizumab state-of-the-art update.

IntroductionEmicizumab is a bispecific monoclonal antibody developed to address the unmet needs of clotting factor replacement therapy and has become the benchmark for optimal prophylaxis in managing patients with haemophilia A with inhibitors. We describe the emicizumab rollout and pharmacokinetic strategies and their use in paediatric patients.MethodsThe evolving real‐world experience in using emicizumab has confirmed its safety, efficacy and pharmacokinetic profile in paediatric, adolescent and adult patients receiving emicizumab at various prophylactic dosing regimens. The emicizumab current global rollout includes over 100 countries with 29 low to middle‐income countries accessing emicizumab through the World Federation of Haemophilia (WFH) Humanitarian Aid Program. The diversity of emicizumab dosing and pharmacokinetic tools such as the Calibra® and the WAPPS‐Hemo platforms make it possible to achieve prophylaxis goals in line with the WFH Haemophilia treatment guidelines recommendations, with minimal drug wastage. The emerging experience from long term clinical trials and long‐term real‐world follow‐up confirm the safety, efficacy, and pharmacokinetic profile of emicizumab in paediatric haemophilia A patients. A few questions, including inhibitor recurrence, concurrent use of emicizumab with various replacement therapies and inhibitor eradication, are being addressed through multiple ongoing clinical studies.ConclusionThe current global rollout of emicizumab is remarkable, and versatile dosing regimens and evolving pharmacokinetic tools such as the Calibra® and WAPPS‐Hemo platforms make it a treatment choice available also for pharmacokinetic guided personalised treatment. Data from paediatric studies are consistent with those seen in adolescent and adult Haemophilia A.

Resection and total femoral prosthesis reconstruction treatment of massive femoral hemophilic pseudotumor: A case report.

Introduction and importanceHemophilic pseudotumor is a rare and serious complication of hemophilia, often occurring in the long bones and muscle tissue of the extremities, with an incidence of about 1–2%. However, there is no effective surgical treatment for massive hemophilia pseudotumors of the extremities. Therefore, we would like to report this case to provide new ideas and methods for the treatment of massive hemophilic pseudotumors of the extremities through resection and total femoral prosthesis reconstruction.Case presentationAfter admission, the patient first underwent a month-long coagulation factor replacement therapy to maintain the patient's factor IX level at approximately 100%. Then, extensive resection of hemophilic pseudotumor and total femoral prosthesis reconstruction was performed in collaboration with several departments, and we collected the patient's preoperative, intraoperative and postoperative data for analysis.Clinical discussionAfter coagulation factor replacement therapy the patient's factor IX level was significantly increased and approached 100%. The surgical procedure was uneventful, and postoperative X-ray suggested a well-positioned prosthesis with postoperative pathological features consistent with a hemophilic pseudotumor. The patient was able to move around with the assistance of a walker 14 days after surgery, and there was no recurrence after one year of follow-up.ConclusionFor treat the massive hemophilic pseudotumors of the extremities, extensive resection and prosthetic replacement with coagulation factor replacement therapy is a safe and effective treatment that can significantly improve the prognosis and quality of life of patients.

Lower Adherence to Clotting Factor Replacement Therapy in Patients with Haemophilia Associated with More Depressive Symptoms: Two Centers Cross-Sectional Study

Lower Adherence to Clotting Factor Replacement Therapy in Patients with Haemophilia Associated with More Depressive Symptoms: Two Centers Cross-Sectional Study

Paciente hemofílico com doença arterial coronariana em pré-operatório de transplante de fígado

Hemophilia A and B, although rare, are the most common clotting disorders in humans. With the advent of prophylaxis and improved clotting factor replacement therapy, patients with hemophilia have reached a life expectancy similar to that of the general population, leading to an increase in cardiovascular risk factors and consequently, coronary artery disease. The need for antiplatelet and anticoagulant therapy in these patients further increases the bleeding tendency. Evidence and recommendations for these patients in procedures such as percutaneous coronary intervention are scarce in the literature.

Effectiveness of clotting factor replacement therapy after antivenom treatment on coagulopathic envenomation following green pit viper bites: a retrospective observational study

BackgroundGreen pit vipers (GPVs), namely Trimeresurus albolabris and Trimeresurus stejnegeri accounts for most snakebites in Southern China. Green pit viper venom contains thrombin-like enzymes, resulting in defibrination syndrome. Using of clotting factor replacement after antivenom administration is controversial. The objective of this study was to investigate the effects of clotting factor replacement in coagulopathic patients with T. albolabris and T. stejnegeri bites after antivenom administration.MethodsWe retrospectively reviewed 123 patients who were bitten by T. albolabris and T. stejnegeri and were admitted to the Emergency Department of a hospital in Guangzhou, Southern China, from 2013 to 2019. Recovery of prothrombin time (PT) and fibrinogen level were compared among (1) fresh-frozen plasma (FFP) group; (2) cryoprecipitate (cryo) group; (3) FFP and cryo group; and (4) control group after antivenom administration.ResultsThe incidence of coagulopathy was 31%. Persistent and late coagulopathy were the most common patterns among four groups. The median reduction in PT was 20.1 ± 31.2 s for FFP and cryo group. The median increase in fibrinogen level was very small: 0.05 ± 0.20 g/L for FFP group, 0.09 ± 0.37 g/L for cryo group and 0.07 ± 0.31 g/L for FFP and cryo group, respectively. The percentage of unimproved PT was markedly higher in the FFP and cryo group than the control group (P = 0.01 by log-rank test, P = 0.02 by Gehan-Breslow-Wilcoxon test). The percentage of unimproved fibrinogen level tended to be worse in the FFP and cryo group than the control group, but the different was marginal (P = 0.05 by Gehan-Breslow-Wilcoxon test, P = 0.07 by log-rank test). A total of 7.8% (7/90) of the patients in the clotting factor replacement groups developed anaphylaxis and heart failure.ConclusionThere is no improvement in coagulopathy profile in patients with T. albolabris and T. stejnegeri bites who received clotting factor replacement after antivenom administration. But the results from GPVs may not be generalized to other species of venomous snakes.

Immune tolerance induction in the era of emicizumab - still the first choice for patients with haemophilia A and inhibitors?

The development of inhibitory antibodies is a severe complication of clotting factor replacement therapy in patients with severe haemophilia A (HA). Current World Federation of Hemophilia (WFH) guidelines for haemophilia care indicate that eradication of inhibitors is best achieved through immune tolerance induction (ITI) therapy. The European Collaborative Haemophilia Network conducted a survey to determine whether ITI is still used in the routine management of patients with HA, and whether the availability of emicizumab prophylaxis has influenced treatment decisions. The survey was conducted in late 2020/early 2021 in 18 centres representing 17 countries in the Europe/Middle East region treating a total of 4955 patients, and included sections specific to patient and centre demographics, treatment protocols (both ITI and prophylactic), inhibitor development and initiation of ITI, treatment success, and the incidence of adverse events. While our results indicate that ITI can still be considered a mainstay of treatment for patients with HA with inhibitors, less than daily dosing of ITI in combination with emicizumab prophylaxis is becoming commonplace across the spectrum of disease severity, with initiation being guided by bleeding patterns. The most frequently cited reasons for not initiating emicizumab prophylaxis were availability or reimbursement issues. ITI remains a mainstay for haemophilia treatment of patients with HA with inhibitors, but emicizumab has become a preferred first-line approach to protect against bleeds and represents an alternative to burdensome ITI in certain patient groups.

Prophylaxis in children with haemophilia in an evolving treatment landscape.

For children with haemophilia, early initiation of prophylaxis is crucial to prevent life-threatening bleeds and maintain joint health throughout life. Options for prophylaxis have recently increased from replacement therapy with standard or extended half-life coagulation factor products to include other haemostasis products, such as the non-replacement therapy emicizumab. To review key factors that determine the choice of prophylaxis in young children. Key clinical questions on the implementation of prophylaxis for haemophilia in children were identified and PubMed was searched for evidence supporting guidance on the implementation of prophylaxis. The results of the literature search and the practical experience of the authors were used to build consensus on when to start prophylaxis, the pros and cons of the products available to guide the choice of product, and practical aspects of starting prophylaxis to guide the choice of regimen. In this era of increasing therapeutic choices, available information about the range of treatment options must be considered when initiating prophylaxis in young children. Parents or care givers must be sufficiently informed to allow informed shared decision making. Although plentiful data and clinical experience have been gathered on prophylaxis with clotting factor replacement therapy, its use in young children brings practical challenges, such as the need for intravenous administration. In contrast, our relatively brief experience and limited data with subcutaneously administered non-replacement therapy (i.e., emicizumab) in this patient group imply that starting emicizumab prophylaxis in young children requires careful consideration, despite the more convenient route of administration.

Reprogrammed CD4+ T Cells That Express FoxP3+ Effectively Control Inhibitory Antibody Development in Hemophilic Mice

Introduction: CD4+CD25+FoxP3+ regulatory T cells (Treg) can suppress a range of immune functions, including the activation and proliferation of conventional CD4+ T cells. Inhibitory antibody development to clotting factor replacement therapy in the X-linked disorder hemophilia is dependent on T cell help. Therefore, Tregs play a crucial role in tolerance to the therapeutic replacement protein. Adoptive immunotherapy with autologous or donor Tregs have shown promise in several clinical trials for autoimmune disorders and in transplant conditions. However, antigen-specific Tregs are extremely rare and difficult to isolate. Ectopic overexpression of FoxP3 in conventional CD4+ T cells from hemophilia patients can potentially create a large pool of Tregs, enriched for antigen specificity. Here we show that adoptive immunotherapy with FoxP3 overexpressing CD4+ T cells can control immune responses to clotting factor replacement therapy in a mouse model of hemophilia A. Methods:BALB/c F8 e16−/− hemophilia A mice were repeatedly injected with recombinant B-domain deleted (BDD) FVIII to generate high inhibitory antibody (inhibitor) titers and FVIII-specific T cells. CD4+CD25- conventional T cells were isolated and transduced with pMIGR-eGFP retroviral vector encoding the murine FoxP3 gene (pMIGR-FoxP3-eGFP). 2x106 GFP+ FoxP3+ sorted cells were adoptively transferred into F8 e16−/− mice, and recipients were challenged 1 day later with weekly IV injections of 1 IU BDD-FVIII for 4 weeks. Plasma was tested for inhibitor/antibody formation at 4 and 8 weeks using the Bethesda assay and ELISA. Furthermore, mice with pre-existing inhibitors received adoptive transfer of a single or multiple doses of 2x106 FoxP3+ sorted cells to assess inhibitor reversal. FoxP3+GFP+ sorted cells were adoptively transferred into naïve mice and re-isolated 2 days later to test for phenotypic marker expression, namely CD25, FoxP3, GITR, CD39, CTLA-4, Neuropilin-1, TNFR2, CD127, and CD62L. Antigen-specific suppressive activity was assayed in vitro using FoxP3 transduced CD4+ T cells from transgenic DO11.10 Rag-/- mice with TCR specificity for ovalbumin (OVA). The proliferation of CellTrace Violet labeled DO11.10 Rag-/- responder splenocyteswas calculated in response to stimulation with OVA323-339 peptide. Results:Retroviral transduction of CD4+CD25- cells with the murine FoxP3 gene resulted in a population of cells that phenotypically resemble peripheral Tregs. FoxP3 transduced Tregs underwent antigen specific proliferation in vivo and stably maintained FoxP3 expression in proliferating cells. FoxP3 transduced OVA-specific CD4+ T from DO11.10 Rag-/- mice suppressed the proliferation of transgenic responder cells in response to OVA323-339 peptide antigen. CD4+ T cells transduced with empty vector or freshly isolated natural Tregs were unable to suppress proliferation. Adoptively transferred FoxP3 transduced cells from BDD-FVIII immunized donor mice completely prevented the induction of inhibitors following challenge with repeat injections of BDD-FVIII. FoxP3 transduction of naïve donors (i.e. not FVIII antigen-experienced) were inferior in suppressing inhibitor formation. We are currently testing for reversal / reduction of established inhibitory antibodies with a single or repeat doses of FoxP3 transduced BDD-FVIII immunized donor mice. Conclusions and future directions: Treg based therapies using ex vivo expanded recipient-derived Tregs are currently in clinical trials as primary or adjunct treatment for allograft rejection and autoimmune disease. Here, forced expression of FoxP3 into antigen-specific conventional CD4+ T cells by retroviral gene transfer resulted in a population of antigen-specific cells with phenotypic characteristics of Tregs. These cells effectively suppressed formation of inhibitors in BALB/c F8 e16−/− hemophilia A mice for a sustained 2-month period. We will further explore the mechanism of suppression, including the possibility of endogenous Treg induction via infectious tolerance. DisclosuresBiswas:Bayer Hemophilia Awards Program: Research Funding. Herzog:Novo Nordisk: Research Funding; Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties.

Trends in coagulation factor replacement therapy and medical costs in patients with haemophilia in Taiwan: A population-based, 15-year analysis.

Taiwan's National Health Insurance Program approved reimbursement of prophylactic coagulation factor replacement therapy (CFRT) for patients with haemophilia (PWH) in 2014. To examine 15-year trends and the impact of reimbursement for prophylactic CFRT on its utilization and related medical costs for PWH. We analysed Taiwan's National Health Insurance Database from 2003 to 2017. We included patients with haemophilia A (PWHA) or B (PWHB) receiving coagulating factor. Female patients were excluded because of small sample size. We analysed annual consumption of CFRT units and medical costs. High proportion of days covered (PDC) with CFRT served as an indicator for prophylactic treatment since it reflects routine use of CFRT. We applied interrupted time series analysis (ITSA) to evaluate the impact of reimbursement for prophylactic CFRT on usage patterns and medical costs. We included 896 male PWHA and 181 male PWHB, with 38.1% and 37.0% aged under 18years, respectively. By ITSA, we found the trends in coagulation factor consumption and PDC significantly increased after reimbursement for prophylactic CFRT in both PWHA and PWHB (p values for trend change <0.05). The overall medical costs per patient increased with increasing consumption of coagulation factor; however, ITSA revealed non-CFRT cost decreased after reimbursement of prophylactic CFRT for both PWHA and PWHB (p values <.05). Reimbursement for prophylactic CFRT facilitated growth in rates of prophylactic CFRT and increased related costs, but curbed rising non-CFRT costs. These findings provide strong grounds for future cost-effectiveness studies to leverage prophylactic CFRT for its therapeutic benefits.

Orthopedic treatment of musculoskeletal disorders in hemophilic patients

目的探讨血友病性骨骼肌肉病变的外科手术治疗方法及围手术期凝血因子消耗情况。方法回顾性分析1996年1月至2019年12月在中国医学科学院北京协和医院骨科因骨骼肌肉病变接受手术治疗的261例血友病患者。根据手术创伤大小选择凝血因子替代治疗方案。记录病变的临床表现、手术种类、围手术期凝血因子消耗、输血率及再手术情况，对比单次麻醉下单部位手术与多部位手术的凝血因子消耗及费用。结果261例患者均为男性，平均年龄为30.6（6~65）岁。血友病A 235例，血友病B 26例；重型（因子活性<1%）141例，中间型（因子活性1%~5%）91例，轻型（因子活性>5%）29例。共施行手术338例次（关节内出血及关节炎226例次、血友病并发肌腱肌膜出血挛缩61例次、血友病假瘤45例次、其他6例次）。338例手术中，大型手术270例（79.9%），关节置换203例（60.1%）。因手术部位病变复发再手术14例，再致残率为4.2%。共55例（21.1%）患者在围手术期接受红细胞输注。血友病A患者术前FⅧ的平均用量为（44.4±8.1）IU/kg，术后2周内FⅧ总用量平均为（647±177）IU/kg（40 962 IU）。术后3个月康复期内，6例（2.3%）患者发生手术部位再出血。7例患者术后检出FⅧ抑制物，平均抑制物水平为（13.7±11.2）BU/ml。单次麻醉下多关节手术组（68例）与单关节手术组凝血因子消耗量及费用差异无统计学意义。结论针对血友病性骨骼肌肉病变，在合理补充凝血因子条件下，根据病变的具体情况选择合适的手术治疗方案是有效的缓解症状的方法。单次麻醉及凝血因子替代治疗下进行多关节手术不增加凝血因子消耗，具有更佳的成本效益。

Risk of deep venous thrombosis after total knee arthroplasty in patients with haemophilia A

Venous thromboprophylaxis remains controversial in patients with haemophilia undergoing major orthopaedic surgery, due to clotting factor replacement therapy to correct the patient's inherent haemostatic defect during procedure, at least in theory. We have previously reported that deep venous thrombosis (DVT) was not detected on ultrasonography (US) after total knee arthroplasty (TKA) in 36 Japanese patients with haemophilia. To evaluate the prevalence of DVT after TKA among haemophilic A patients using not only US, but also contrast-enhanced CT prospectively. This study enrolled 11 TKAs in 11 Japanese patients with haemophilia A not having any history of inhibitor. A pneumatic compression device was used from the beginning of the operation until the patient could perform standing exercises (day 2). US of the lower extremities was performed before and after surgery (day 2) to detect DVT. Contrast-enhanced CT was performed after surgery (day 7) to detect VTE. D-dimer was measured at pre- and postoperative time. Mean (±standard deviation) age at the time of operation was 50.5±12.3years. DVT was not found on either pre- or postoperative examinations by US, but contrast-enhanced CT was detected DVT in two cases. No patients showed clinical signs for VTE during hospitalization, and no additional treatment for VTE. No episodes of unexpected bleeding were encountered. Contrast-enhanced CT detected DVT in 18% of haemophilia A patients who underwent TKA despite no detection of DVT on US. We must consider how to prevent and treat VTE in patients with haemophilia scheduled to undergo orthopaedic surgery.