FVIII Inhibitor Titer Research Articles

Evaluating clinically translatable conditioning for platelet gene therapy in murine hemophilia A with inhibitors

BackgroundPlatelet gene therapy is effective in hemophilia A (HA) mice even with inhibitors. Fludarabine (Flu), along with busulfan (Bu) or melphalan (Mel), preconditioning has been shown to be highly effective for hematopoietic stem cell transplantation in the clinic. ObjectivesTo evaluate the efficacy of Bu–Flu and Mel–Flu preconditioning in platelet gene therapy of HA with inhibitors. MethodsBu–Flu and Mel–Flu were used to condition HA mice preimmunized with recombinant human factor (F)VIII. An optimal 660 centigray total body irradiation was used as a control regimen in parallel. Platelet–FVIII expression was introduced by transplantation of 2bF8 lentivirus (LV)-transduced hematopoietic stem cells. Animals were analyzed by fluorescence-activated cell sorting, quantitative polymerase chain reaction, FVIII assays, and tail bleeding tests. ResultsBu–Flu, but not Mel–Flu, enabled successful 2bF8 gene therapy. All recipients achieved >55% chimerism post hematopoietic stem cell transplantation in both Bu–Flu and 660 centigray groups, with comparable copy numbers of 2bF8 cassette and the platelet–FVIII levels. The bleeding phenotype was rescued in 2bF8LV-transduced recipients. FVIII inhibitor titers declined with time, with comparable disappearance time of inhibitors between the 2 groups. When animals were rechallenged with recombinant human FVIII after the titers dropped to undetectable levels, no inhibitors were detected in 2bF8LV-transduced recipients. In contrast, all untransduced transplanted control mice produced inhibitors. These data demonstrate that immune tolerance was established in 2bF8LV-transduced primed HA mice under Bu–Flu conditioning. ConclusionBu–Flu preconditioning allows for successfully introducing platelet–FVIII expression to restore hemostasis and induce immune tolerance in primed HA mice, suggesting that this approach is a promising clinically translatable strategy for gene therapy of HA with inhibitors.

AKATSUKI 48-Week Interim Analysis: Emicizumab and Immune Tolerance Induction in People with Hemophilia A and Factor VIII Inhibitors

Background: Immune tolerance induction (ITI) therapy, for the eradication of factor (F)VIII inhibitors, is indicated for people with hemophilia A (PwHA) with FVIII inhibitors. There are limited data regarding the safety of emicizumab treatment in combination with ITI, particularly regarding thrombotic events (TEs). This study, conducted in Japan, aims to evaluate the safety profile of emicizumab during and immediately after ITI. Methods: AKATSUKI is a Phase IV, open-label, non-randomized, interventional, multicenter study; the methods have been previously published (Matsushita et al. BMJ Open 2022). PwHA with FVIII inhibitors received an approved dose of emicizumab and ITI therapy. Any standard half-life (SHL) FVIII concentrate could be used for ITI, with a dosing regimen of 50IU/kg, three times a week; when using an extended half-life (EHL) FVIII concentrate, 50IU/kg twice a week was permitted. Those who achieved ITI partial success received prophylaxis with emicizumab and a FVIII concentrate starting from the date of their next study visit following investigator confirmation of partial success. Any FVIII concentrate could be used for post-ITI maintenance therapy, given as 50IU/kg once a week for the first 24 weeks followed by 50IU/kg once every 2 weeks for the next 24 weeks, followed by discontinuation of the FVIII concentrate at the investigator's discretion. The primary endpoint was evaluation of adverse events (AEs), with a key focus on TEs, and abnormal laboratory values over time during and immediately after ITI. The main secondary endpoints included number of treated bleeds and the proportion of participants meeting ITI success criteria. Partial success was achieved on the same date that the participant's blood sample showed normal FVIII recovery in addition to the participant receiving a negative FVIII inhibitor titer. Normalized FVIII recovery was defined as a FVIII recovery rate of ≥66% of the predicted value on two consecutive occasions ≥2 weeks apart, and a negative FVIII inhibitor titer was defined as achieving FVIII inhibitor levels of <0.6 BU/mL on two consecutive occasions ≥2 weeks apart. Results: Twelve participants, all of whom were male (median [range] age 2.5 [1-54] years) were included in this interim analysis (evaluation period, median [range]: 437.5 days [224-749]. Data cut-off date: December 22, 2022). Prior to the study, participants had received emicizumab (n=11), ITI (n=9), or both (n=7), and the median (range) duration of ITI in combination with emicizumab was 203.0 (7-1,002) days. Three participants used a SHL FVIII product for their ITI treatment regimen, and nine participants used an EHL FVIII product. Additional baseline characteristics are presented in Table 1. Overall, 30 AEs occurred, none of which were considered emicizumab- or FVIII concentrate-related. Two participants each experienced one serious AE: one case of pneumonia, initially caused by transmission of a viral infection, and one wound hemorrhage, which was recorded as accidental; both recovered. No participants experienced a TE. Three participants reported a total of 10 treated bleeds, all of which were categorized as traumatic and were treated with recombinant activated FVII. At data cut-off, one participant had achieved partial ITI success, having reached a negative inhibitor titer at Week 5 and normalized FVIII recovery by Week 17. This participant was then treated with emicizumab and FVIII concentrate (50IU/kg once a week). No bleeds or AEs were reported for this participant during the observational period. The changes in FVIII inhibitor titer across the study for all participants are presented in Figure 1. Conclusions: At this 48-week interim analysis, no new safety concerns or TEs were reported in PwHA receiving emicizumab and ITI. This study continues to evaluate the safety of emicizumab in conjunction with, and following, ITI therapy in PwHA with FVIII inhibitors.

Single-Dose Rituximab Plus Glucocorticoid Versus Cyclophosphamide Plus Glucocorticoid in Patients with Newly Diagnosed Acquired Hemophilia a: A Multicenter, Open-Label, Randomized Clinical Trial

Introduction Cyclophosphamide plus glucocorticoid is a first-line immunosuppressive therapy for acquired hemophilia A (AHA), a serious bleeding disorder, but brings risks of myelosuppression, secondary tumor, and reproductive toxicity. Rituximab plus glucocorticoid therapy shows comparable efficacy but lacks results from randomized controlled trials. We aimed to establish whether the single-dose rituximab regimen was non-inferior to the cyclophosphamide regimen. Methods This study was the first noninferiority, multicenter, open-label, randomized controlled clinical trial comparing these two combination therapies in AHA patients (ClinicalTrails.gov number, NCT03384277). Adult patients (18-80 years) with newly diagnosed AHA from five centers in China were randomly assigned 1:1 to receive glucocorticoid (methylprednisolone 0.8 mg/kg per day for the first 3 weeks and then tapered) plus single-dose rituximab (375 mg/m 2) or plus cyclophosphamide (2 mg/kg per day until inhibitor becomes negative, for a maximum of 5 weeks). The primary outcome was complete remission (CR, defined as FVIII activity ≥ 50 IU/dL, FVIII inhibitor undetectable, immunosuppression tapered and no bleeding for 24 hours without bypassing agents) rate measured within 8 weeks. The noninferiority margin was an absolute difference of 20%. The secondary outcomes included partial remission (PR) rate, time to PR, time to CR, time for inhibitor titer to decrease by 50% of baseline, relapse rate, time to relapse and so on. Safety was assessed in all patients. Results Between December 29, 2017 and June 9, 2022, 67 patients were screened, of whom 63 (33 female and 30 male) were assigned: 31 to single-dose rituximab group and 32 to cyclophosphamide group. The baseline characteristics of these patients were balanced between the single-dose rituximab group and cyclophosphamide group. Twenty-four (77.4%) patients in the rituximab group and twenty-two (68.8%) patients in the cyclophosphamide group achieved CR, which showed the noninferiority of the single-dose rituximab-based regimen (absolute difference=-8.67%, lower limit of the 95% confidence interval=-13.11%; P noninferiority=0.005). PR was observed in 25 (80.6%) of 31 patients in the single-dose rituximab group and 26 (81.3%) of 32 patients in the cyclophosphamide group (P=0.95). The median time to PR was similar between two groups (23.0 days for rituximab group vs. 25.5 days for cyclophosphamide group, P=0.47). The median time to CR was 8 days shorter in the single-dose rituximab group than in the cyclophosphamide group, but with no statistically significant difference (28.0 days vs. 36.0 days, P=0.41). The median time for inhibitor titer to decrease by 50% of baseline also showed a shorter trend in the single-dose rituximab plus glucocorticoid group (8.0 days vs. 11.0 days, P=0.39). Subgroup analysis showed patients with high FVIII inhibitor titer (>20 BU/ml) were less likely and slower to achieve both CR and PR. The percentage of patients who experienced treatment-related adverse events (TRAEs) or grade 3 or 4 TRAEs were comparable between the rituximab and cyclophosphamide groups (58.1% vs. 37.5%, P=0.10 and 12.9% vs. 6.3%, P=0.43 respectively). Conclusion Single-dose rituximab plus glucocorticoid regimen showed similar efficacy and safety in this trial, without a reported risk of secondary malignancies or reproductive toxicity seen in cyclophosphamide, it might be recommended as a first-line therapy for AHA, especially in China where there is a young age trend in AHA patients.

The Tfh/Tfr Pathway Is Pivotal in FVIII Inhibitor Development in Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. Our previous study demonstrated that the induction of activated follicular helper T (Tfh) cells in FVIII null mice is critical for FVIII inhibitor development. Follicular regulatory T (Tfr) cells are a specialized subset of Foxp3 + T cells identified in the germinal center (GC) that can modulate Tfh cell activation of B cells and antibody development. However, the role of Tfr cells in controlling anti-FVIII inhibitor development in vivo is still unclear. Here, we explored the activation and induction of Tfr cells and their functions in anti-FVIII inhibitor development in FVIII-immunized mice. We first characterized the features of activated Tfr cells in the spleen of immunized FVIII null mice by flow cytometry analysis. Activated Tfr cells were defined as CD4 +Foxp3 +CD44 +CD62L -CXCR5 +PD-1 +. Tfr cells express a high level of transcription factor BCL6 and costimulatory receptor ICOS. Activated Tfr cells also express a high level of costimulatory receptor CTLA4, which is similar to non-Tfr effector Treg cells, but CD25 expression is low on Tfr cells. FVIII immunization increases the frequency and the absolute number of activated Tfr cells in the spleen of mice that produce inhibitors. The percentage of activated CXCR5 hiPD-1 hi Tfr cells within the CD4 population significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (0.88 ± 0.2 %, 0.33 ± 0.16 %, and 0.36 ± 0.2%, respectively). The Tfh/Tfr ratio also significantly increased in FVIII inhibitor-producing mice, suggesting a preferential expansion of Tfh cells relative to Tfr cells in inhibitor-producing mice. Interestingly, similar to Tfh cells as we reported before, the emergence of Tfr cells correlated with titers of anti-FVIII inhibitors in FVIII immunized mice. This indicates that a functional relationship exists between Tfr cell activation and FVIII inhibitor production after FVIII immunization. Using Tfr deficient Foxp3-Cre Bcl6 fl/fl (BCL6FC) mice, we determined the role of Tfr cells in inhibitor generation in vivo. Loss of Tfr cells led to significantly decreased FVIII inhibitor levels, with FVIII inhibitor titers in BCL6FC mice markedly lower than in wild-type (WT) mice upon recombinant human full-length FVIII (rhFVIII, Kogenate 200 U/kg/week IV x4) immunization (24 ± 16 and 131 ± 114 BU/ml, respectively). However, the total anti-FVIII IgG titers were similar between the BCL6FC and WT groups, suggesting that Tfr cells help generate neutralizing anti-FVIII antibodies. FVIII immunization could still efficiently induce Tfh cell activation in BCL6FC mice. However, both GC Tfh cells and GC B cells were reduced in FVIII immunized BCL6FC mice, suggesting that Tfr cells promote FVIII-specific GC responses. To further investigate the regulatory function of Tfr cells on FVIII immune responses, mice with phosphatase Pten deficiency in Treg cells (Foxp3-Cre Pten fl/fl [PtenFC]), a model with augmented Treg and Tfr cells, were immunized with rhFVIII and anti-FVIII antibody titers were measured at 1 week after the 4 th weekly immunization. Of note, FVIII inhibitor titers (8.06 ± 8.6 BU/ml) were significantly lower than those in WT controls under the same immunization protocol. There were significant positive correlations between the inhibitor titers and total anti-FVIII IgG in the WT and PtenFC groups but not in the BCL6FC group after rhFVIII immunization. To confirm the important role of the Tfh/Tfr pathway in FVIII inhibitor development, we used CD4-Cre Bcl6 fl/fl (CD4-Bcl6-cKO) mice, a model with Bcl6 deficiency in all CD4 + T cells. As expected, none of the CD4-Bcl6-cKO mice developed detectable levels of FVIII inhibitors, demonstrating that the transcription repressor BCL6 is pivotal for FVIII inhibitor development. In conclusion, we found that FVIII immunization induces Tfr cell activation and expansion. Tfr cells have a dual function in regulating FVIII immune responses in mice, and the Tfh/Tfr pathway is essential for FVIII inhibitor development in hemophilia A mice.

Effect of emicizumab-neutralizing antibodies on activated partial thromboplastin time–based clotting time test results in patients treated with emicizumab

BackgroundEmicizumab is a bispecific humanized monoclonal antibody that shortens the activated partial thromboplastin time (aPTT), making aPTT-based tests unreliable. ObjectivesTo evaluate the efficacy of a mixture of 2 anti-idiotype monoclonal antibodies (anti-emi) in neutralizing emicizumab in samples from persons with hemophilia A treated with emicizumab. MethodsFifty samples from persons with hemophilia A treated with emicizumab were analyzed for aPTT and factor VIII procoagulant activity; FVIII inhibitor titer was measured using Nijmegen-Bethesda assay in 50 plasma samples of additional patients (positive for FVIII inhibitor) treated with emicizumab. FVIII procoagulant activity and inhibitor titer were measured using 1-stage (Actin FS, Siemens) and chromogenic assays with bovine regents (Factor VIII Chromogenic Assay, Siemens). Emicizumab concentration was measured by modified a 1-stage assay calibrated with a drug-specific calibrator (r2 Diagnostics Inc). All the tests were performed on Sysmex CS-2400 (Sysmex) before and after the addition of anti-emi (Chugai Pharmaceutical). ResultsEmicizumab concentrations measured after neutralization were <1.6 μg/mL in all samples. FVIII levels were >480 IU/dL with an aPTT of <30.8 seconds in all samples before neutralization and were <1 IU/dL with an aPTT of >70 seconds after adding anti-emi. FVIII inhibitor resulted in a false negative result in 44 of 50 samples measured with the 1-stage assay before neutralization. A good correlation (r = 0.98) was found between inhibitor titer measured using the chromogenic (insensitive to emicizumab) and 1-stage assays after neutralization. ConclusionThe anti-emi antibodies were shown to completely neutralize emicizumab, making samples pretreated with anti-emi analyzable with the 1-stage assay.

High levels of factor VIII activity in patients with acquired hemophilia A in remission are associated with unusually low coagulation potentials.

Elevated factor VIII activity (FVIII:C) is often observed in patients with acquired hemophilia A (PwAHA) in remission. However, comprehensive coagulation potentials in this patient group remain to be investigated. To evaluate comprehensive coagulation potentials in PwAHA. We investigated coagulation function in eleven PwAHA with high FVIII:C (> 150IU/dL) using thrombin generation assay (TGA) and/or rotational thromboelastometry (ROTEM), and compared findings with results obtained from contrived samples generated by spiking recombinant FVIII. The median FVIII:C and FVIII inhibitor titers during remission in enrolled PwAHA were 206IU/dL and 0.44 BU/mL, respectively. In all patients, lag time and time to peak were either prolonged or normal compared to contrived samples corresponding to their FVIII:C. However, higher values of peak thrombin and endogenous thrombin potentials compared to contrived samples were observed in two patients. ROTEM parameters were within normal ranges in all cases. One patient (FVIII:C 171IU/dL) developed venous thrombosis and pulmonary embolism, but TGA parameters showed low or normal coagulation potential compared to contrived samples corresponding to his FVIII:C. PwAHA with high FVIII:C could exhibit lower coagulation potentials than those corresponding to their FVIII:C.

High Titer Inhibitor-Acquired Hemophilia A Treated With Human FVIII Concentrate in Limited Resource: A Case Report

Acquired hemophilia A (AHA) is a rare condition that affects one in a million people each year, and there are not many diagnostics or therapeutic agents available for treatment due to its rarity. This is a case report of a 61-year-old woman who presented with a spontaneous subcutaneous hematoma and multiple extensive bruises in her extremities. There was no prior history of bleeding disorders, and the laboratory results showed an isolated aPTT prolongation with no correction after mixing studies, and a reduction in FVIII activity level along with a high FVIII inhibitor titer (928BU). Furthermore, the diagnosis of idiopathic AHA was made after other secondary causes had been ruled out, and the patient received human FVIII concentrate instead of bypassing agents due to its availability. The patient still experienced clinical improvement despite using this alternative. AHA is currently managed using both hemostasis agents and inhibitor eradication, and they come with several limitations. Human FVIII concentrate therapy is still an option in situations with limited resources, even though it is not recommended in patients with high inhibitor titer levels.

AKATSUKI study: a prospective, multicentre, phase IV study evaluating the safety of emicizumab under and immediately after immune tolerance induction therapy in persons with congenital haemophilia A with factor VIII inhibitors

IntroductionFor persons with haemophilia A with factor (F) VIII inhibitors (PwHAwI), immune tolerance induction (ITI) therapy is indicated for inhibitor eradication; however, since PwHAwI on ITI were excluded from the...

Feasibility of therapeutic plasma exchange-based combination therapy in the treatment of acquired hemophilia A: A retrospective 6 case series.

Poor availability and a lack of affordability of bypassing agents (recombinant activated factor VII and activated prothrombin complex concentrate) in west China prompted us to investigate an alternative cost-effective combination therapy. We aimed to explore the feasibility of therapeutic plasma exchange (TPE)-based combination therapy in the treatment of acquired hemophilia A (AHA).We retrospectively investigated the clinical features of AHA in 6 patients who were treated with a combination of TPE, corticosteroids, and rituximab in our department for 9 years between January, 2011 and December, 2019.We examined 1 male and 5 female patients. The median age at diagnosis of AHA was 51 years (18–66 years). In all patients, FVIII activity levels were low (median: 1.5%; 1–3%), FVIII inhibitor titers were high (median: 24.5 BU/mL; 13.2–48.6 BU/mL), and activated partial thromboplastin time was markedly prolonged (median: 99.4 s; 60.9–110.1 s). They underwent 2 to 8 cycles of plasma exchange and were given varying combinations of dexamethasone, methylprednisolone, prednisone, and rituximab. After TPE bleeding gradually stopped, and activated partial thromboplastin time decreased. After 3 months of treatment, FVIII inhibitors completely disappeared.TPE when combined with corticosteroids and rituximab, as adjunctive immunosuppressive agents, may be an effective and reliable treatment for AHA. When there is no alternative, intensive first-line treatment including TPE may be lifesaving.

Efficacy and Safety of Third-Generation Full Length Recombinant Human Coagulation Factor VIII in the Standard Prophylactic Treatment of Childhood Patients with Moderate-to-Severe Hemophilia A: A Single Center Experience

Objective To evaluate the number of annual bleeding and annual joint bleeding and safety of third-generation full length recombinant human coagulation factor Ⅷ (ADVATE) in the standard prophylactic treatment of childhood patients with moderate-to-severe hemophilia A. Methods Fifteen children aged from 5 to 18 years with moderate-to-severe hemophilia A were enrolled in the one center, prospective, nonrandom, self-control study from Nov 01,2014 to Nov 30,2016. Children were treated with one-year long-course infusion of ADVATE with a dose of 20 ∼ 40 IU/kg twice-weekly or three times weekly. The numbers of annual bleeding and annual joint bleeding after the prophylaxis treatment were compared with that on-demand. Moreover, any adverse effect and serious adverse reaction related to ADVATE usage were documented during the study. The FⅧ inhibitor titer were measured with modified Nijmegen method before and after the prophylaxis every 3 to 6 months. Results The FⅧ:C for these 15 boys, median age 15, was less than 2.2%, with 8 patient severe type. The number of annual bleeding was reduced from 27.6 on-demand treatment to 2.6 after the prophylaxis treatment(P < 0.001), and the number of annual joint bleeding was also reduced from 11.1 to 0.9(P < 0.001). Seven patients had zero annual joint bleeding and the mean annual bleeding for mild, moderate and severe degree was decreased significantly with zero times for severe degree bleeding after the standard prophylactic treatment. No adverse effect or serious adverse reaction related to ADVATE was recorded during one-year period treatment and FⅧ inhibitor detection was negative. Conclusions Standard prophylactic treatment with ADVATE can effectively decrease the annual bleeding and annual joint bleeding in children with moderate-to-severe hemophilia A. This therapy is safe with FⅧ inhibitor test negative. DisclosuresNo relevant conflicts of interest to declare.

Emicizumab Prophylaxis in Adolescent/Adult Patients with Hemophilia A Previously Receiving Episodic or Prophylactic Bypassing Agent Treatment: Updated Analyses from the HAVEN 1 Study

Emicizumab Prophylaxis in Adolescent/Adult Patients with Hemophilia A Previously Receiving Episodic or Prophylactic Bypassing Agent Treatment: Updated Analyses from the HAVEN 1 Study

Inhibitor Index in the Clot Waveform Analysis-Based Mixing Test Differentiates among Hemophilia A without and with Inhibitors, and Lupus Anticoagulant.

The mixing test is used to identify the pathway to follow-up testing and is also useful for the investigation of lupus anticoagulant (LA) positivity. "To completely correct" indicates clotting factor deficiency, while "to not correct" indicates the presence of a clotting factor inhibitor including LA. "Index of circulation anticoagulant" and/or "percent correction" is used to interpret the results of mixing studies, but it does not accurately differentiate factor inhibitors from LA. To precisely differentiate hemophilia A (HA), HA with inhibitor (HA-inh), and LA using the clot waveform analysis (CWA)-based mixing test. Plasma samples from HA, LA, and HA-inh including acquired HA were incubated with normal plasma in 9:1, 1:1, and 1:9 mix ratios. From activated partial thromboplastin time CWA at 0-minute (immediately) and 12-minute incubation, the ratios of CWA parameters at 12 minutes/0 minute (inhibitor index) were assessed. The inhibitor index values of CWA parameters obtained using the mixing test in a 1:1 ratio demonstrated a significant difference between HA-inh and LA but could not differentiate LA from HA-inh completely. Plasmas used for the mixing tests in 9:1 and 1:9 ratios were able to fully distinguish between HA-inh (>0.5 BU/mL) and LA. These indices significantly correlated with inhibitor titer below 40 BU/mL (r > 0.90), possibly estimating FVIII inhibitor titer from the inhibitor index. Plasmas in HA and LA could be distinguished by mixing in a 1:1 ratio at 0 minute (immediately). The inhibitor index from CWA-based mixing tests with a 12-minute incubation could differentiate among HA, HA-inh, and LA quickly.

First-in-Human Phase 1/2 Clinical Trial of SIG-001, an Innovative Shielded Cell Therapy Platform, for Hemophilia Α

First-in-Human Phase 1/2 Clinical Trial of SIG-001, an Innovative Shielded Cell Therapy Platform, for Hemophilia Α

Routine measurements of factor VIII activity and inhibitor titer in the presence of emicizumab utilizing anti‐idiotype monoclonal antibodies: reply

Essentials Emicizumab (Emi) affects the APTT-based assays of factor (F)VIII activity and inhibitor titer. A mixture of two anti-Emi monoclonal antibodies (mAb) effectively neutralized the Emi activity. Anti-Emi mAbs completely eliminated the influence of Emi on FVIII activity and inhibitor titer. The inclusion of anti-Emi mAbs in routine FVIII assays would be useful for Emi-treated patients. Summary Background Emicizumab is an anti-factor (F)IXa/X bispecific monoclonal antibody (mAb), mimicking the factor (F)VIIIa cofactor activity. Emicizumab does not require activation by thrombin and its shortening effect on the activated partial prothrombin time (APTT) is more pronounced than that of factor (F)VIII. APTT-based FVIII activity (FVIII:C) and FVIII inhibiter titer measurements are influenced by the presence of emicizumab. Aim To establish a reliable APTT-based assay to measure FVIII in the presence of emicizumab. Methods Plasmas from hemophilia A (HA) patients without or with inhibitors were studied using one-stage FVIII:C and Bethesda inhibitor assays. Two recombinant anti-idiotype mAbs to emicizumab (anti-emicizumab mAbs) were prepared, rcAQ8 to anti-FIXa-Fab and rcAJ540 to anti-FX-Fab. Results The combined anti-idiotype mAbs (2000 nm each) eliminated the effects of emicizumab on APTTs of HA plasmas without or with inhibitor by competitive inhibition of antibody binding to FIX(a)/FX(a). Measurements of FVIII coagulation activity in HA plasmas without inhibitor were overestimated in the presence of emicizumab (1 μm = ~150 μg mL-1 ) at all reference levels of FVIII. The addition of anti-emicizumab mAbs to the assay mixtures completely neutralized the emicizumab and facilitated accurate determination of FVIII:C. Anti-FVIII inhibitor titers were undetectable in the presence of emicizumab in HA plasmas with inhibitor or normal plasmas mixed with anti-FVIII neutralizing antibodies. These effects of emicizumab were completely counteracted by the addition of the anti-idiotype mAbs, allowing accurate assessment of inhibitor titers. Conclusion The in vitro inclusion of anti-emicizumab mAbs in the standard one-stage coagulation assays prevented interference by emicizumab and enabled accurate measurements of FVIII:C and inhibitor titers.

Recombinant Porcine Factor VIII Use in Bleed Treatment in Non-Severe Hemophilia a Inhibitor Patients: Dosing Strategies and Efficacy

Introduction and Rationale:The development of alloantibodies to factor VIII remains a challenging and significant complication in the treatment of patients with congenital hemophilia A (HA). Inhibitor development is more common in severe HA than in non-severe HA (NSHA). Incidence of inhibitors in NSHA is approximately 3-13%, and even with attempted immune tolerance induction and immunosuppression, inhibitors have been reported to persist long-term in about 40% of NSHA patients (Hay 1998). Inhibitors in patients with NSHA can have variable kinetics, causing clinical characteristics similar to patients with acquired hemophilia A (AHA), with observable type 2 kinetics and inhibition of exogenous and endogenous FVIII activity (d'Oiron et al. 2006). Optimal treatment regimens have yet to be established for these patients, in terms of inhibitor eradication and choice of agent for control of bleeding episodes, with known thrombotic side effects when using bypassing agents, including in pediatric patients (d'Oiron et al. 2006).It has been observed as well that the efficacy of bypassing agents, such as activated prothrombin complex concentrates (aPCC) and recombinant activated factor VII (rFVIII), is diminished in the presence of an inhibitor (Rocino et al. 2017). Challenge with aPCC or rFVIII can also induce inhibitor recurrence in patients who have had previous resolution (Giuffrida et al. 2008).Recombinant porcine FVIII (rpFVIII) is an alternative therapy, which by virtue of its incomplete homology in the A2 and C2 domains of the FVIII molecule, has decreased reactivity with anti-human FVIII (hFVIII) inhibitors (Mulliez et al. 2014). rpFVIII has demonstrated efficacy in patients with severe HA with inhibitors, as well as in acquired HA (AHA) (Kempton et al. 2012; Kruse-Jarres et al. 2015). However, experience is limited for use in patients with NSHA and inhibitors. We report successful bleed treatment with rpFVIII of 3 adult patients and 1 child with NSHA who developed inhibitors at the University of North Carolina, with excellent hemostatic efficacy and no adverse treatment-related effects.Methods:We analyzed clinical, pharmacy, and laboratory data from 4 patients with NSHA who developed inhibitors treated with rpFVIII after failure of or contraindication to bypassing agents at the University of North Carolina. Investigational review board approval was obtained for our data collection and analysis.Results:Average baseline FVIII activity was 9.5%, with 3 of our 4 patients having a FVIII activity of <1 after inhibitor development. Mean anti-human FVIII inhibitor titer (hBIA) was 83 BU, ranging from 0.8 to 306 BU. Mean cumulative dose of rpFVIII used in the first 24 hours was 735 U/kg, but only 163 U/kg in our adult patients. Mean number of infusions used in the first 24 hours was 2.3 in our adult patients, and the pediatric patient required 18 infusions in the first 24 hours. Mean peak FVIII recovery was 153%. Hemostasis was achieved in all 4 patients with no treatment-related adverse effects, despite 3 of 4 patients developing a detectable anti-porcine FVIII inhibitor titer (pBIA).Conclusions:rpFVIII appears to be a safe and effective therapy in patients with NSHA who develop inhibitors. Our patients treated with immunosuppression in addition to rpFVIII, similar to how we treat patients with AHA. Of note, we achieved effective hemostasis in our 3 adult patients at much lower than FDA-recommended doses for AHA by using our previously established dosing algorithm for rpFVIII (Martin et al. 2016; Fig 1). Our pediatric patient required much more rpFVIII for effective hemostasis. This patient also had a much higher hBIA, and was being treated after the failure of multiple bypassing agents. However, once it seemed inhibitor binding sites were saturated after frequent higher rpFVIII doses, FVIII recovery was achieved, with concomitant decrease in doses required. FVIII activity was an effective means of monitoring efficacy of rpFVIII use, as in severe HA and AHA. One adult patient in particular had a very good clinical response to rpFVIII, requiring only 1 dose in the first 24 hours, with doses as low as 19 U/kg given daily required for bleeding control. [Display omitted] DisclosuresAbajas:CSL Limited: Honoraria; Bayer: Honoraria. Moll:Stago Diagnostics: Consultancy. Ma:CSL: Consultancy; Novo Nordisk: Consultancy; Shire: Honoraria, Research Funding; CVS: Honoraria.

A Standardized Kit for a Chromogenic Modified Nijmegen-Bethesda Assay: Repeatability, Reproducibility, and Analytical Sensitivity

Antibody-based inhibitor response to Factor VIII replacement therapy is one of the most common complications in the treatment of persons with hemophilia A (PwHA). Accordingly, accurate quantification of FVIII inhibitor antibody titer is essential in guiding patient treatment. Testing for FVIII inhibitors is not standardized; typically, FVIII inhibitor titers are measured using non-standardized LDTs (Laboratory Developed Tests). These LDTs vary in the processing of patient plasma, the reagents used to perform the assay, and the method of FVIII activity measurement.Here we report the development of a standardized set of reagents to perform a chromogenic modified Nijmegen-Bethesda assay (MNBA). The kit consists of imidazole-buffered pooled normal plasma (IB-PNP) with a consistent normal level of FVIII activity, and an imidazole-buffered bovine serum albumin solution (IB-BSA) as a diluent. Also included in the kit are a positive FVIII inhibitor plasma control (1.2 - 1.8 BU/mL) and an inhibitor-free negative plasma control.To perform the assay, test plasma was heat inactivated at 56 °C for 30 minutes to minimize residual FVIII activity. The inactivated plasma was then centrifuged (2700 x g,5 min.) and the supernatant used to make a series of two-fold serial dilutions with IB-BSA. The dilutions were each mixed with an equal volume of IB-PNP to form test mixes and incubated in a 37 °C water bath to allow time for the inhibitor antibodies from the patient plasma to inactivate FVIII in the IB-PNP. After two hours, the reaction was halted by cooling the test mixes in an ice bath. The FVIII activity of the test mixes was then measured using a Siemens FVIII Chromogenic Assay on a Siemens BCS XP analyzer. The residual FVIII activity of each test mix was calculated by comparing its FVIII activity to the activity of a control mix containing no test plasma (1:1 mixture of IB-BSA and IB-PNP).Here we report the use of this kit to perform a comprehensive study examining the repeatability, reproducibility, and analytical sensitivity of a chromogenic MNBA performed according to CLSI guidelines (CLSI EP05 and EP17). Inhibitor-negative and inhibitor-positive plasma from PwHA were combined to yield a panel of test plasmas at four different levels of inhibitor: below cutoff level (~0.3 BU/mL), low positive (~1.2 BU/mL), mid positive (~5 BU/mL) and a high positive (~8 BU/mL). To assess the repeatability of the assay, we measured each of these test plasmas using three lots of MNBA kit and one lot of FVIII chromogenic kit for a total of 240 titer determinations (3 lots × 20 days × 2 runs × 2 replicates). Inhibitor measurements of the panel of test plasmas showed within-lot precision of less than 10% (CV), and the kit controls were identified correctly each time. The reproducibility of the assay was determined in a study conducted across three different laboratories using different BCS XP analyzers and operators but the same panel of test plasmas. The limit of blank (LoB) and the limit of detection (LoD) of the assay were also determined using multiple low titer plasmas from unique donors. The assay system can reliably distinguish inhibitor titers as low as 0.2 BU/mL (LoD) in plasma from PwHA with no history of inhibitors.The chromogenic MNBA kit shows potential for labs seeking repeatable and reproducible FVIII inhibitor measurement that can otherwise vary significantly within or between labs. Standardization of reagents and protocol yields consistent results and is suitable for multi-center inhibitor studies in PwHA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The use of thromboelastography, thrombin generation test and clotting tests to evaluate the efficacy of hemostatic therapy with recombinant activated factor VII in hemophilia patients with inhibitor

Introduction . The Problem of the use of recombinant activated factor VII (rFVIIa) in hemophilia patients with inhibitors is the complexity of the laboratory evaluation of the therapy. After rFVIIa administration standard clothing tests are changing, however, are not normalized. The aim of this study was to investigate the possibility of using standard clothing tests (activated partial thromboplastin time (APTT) and prothrombin time (PT)) to assess the efficiency of hemostatic therapy with rFVIIa in hemophilia patients with inhibitors. The aim of this work is to investigate the possibility of using standard clothing tests (activated partial thromboplastin time (APTT), prothrombin time (PT)) to assess the efficiency of hemostatic therapy with rFVIIa in patients with inhibitory hemophilia. Materials and methods. 20 male hemophilia patients with inhibitors were included in the study. At the time of study inclusion none patients had signs of bleeding. Plasma levels of FVIII were < 1 %, FVIII inhibitor titers were from 5 BU/ml to 463 BU/ml. All patients received rFVIIa (Coagil-VII) at a dose of 90 μg/kg. Before the administration of rFVIIa and then in 15, 30 and 60 min, 2 and 24 h the APTT, the PT, the endogenous thrombin potential (ETP) and thromboelastography (TEG) parameters (reaction time – R, maximum amplitude – MA) were evaluated. Results. Before rFVIIa administration all patients had prolonged APTT. 15 min after administration of rFVIIa APTT shortened and remained shorter than baseline level, but 2 times longer than normal ranges during 2 hours. The PT also significantly decreased in 15 min after the rFVIIa administration. Prior to treatment patients had minimal to no clotting detectable by TEG. Administration of rFVIIa led to normalization of TEG traces in the most of the patients in 15 min. Elevated by TEG hemostatic effects persisted for 2 h. The ETP increased in 15 min after rFVIIa administration. This increase of ETP persisted for 60 min. There were strong correlations between R and APTT (r = 0.74; p = 0.001), between MA and APTT (r = 0.70), between PT and R (r = 0.79; p = 0.01), PT and MA (r = 0.76; p = 0.01). There were no correlations between ETP and APTT, and between ETP and R. The shortening of the APTT after rFVIIa administration for 17 s and more or for 22 % and more from the baseline levels were associated with the normalization of R and MA. Changes of the PT poorly allowed to discriminate normal values of TEG. Conclusion . Shortening of the APTT after rFVIIa administration in hemophilia patients with inhibitor for 17 s and more or for 22 % and more from the initial value can be used to assess the hemostatic efficiency of rFVIIa therapy.

The Effect of Inhibitory Antibodies on Acute Treatment of Bleeding Episodes with OΒΙ-1, an Antihemophilic Factor VIII (recombinant), Porcine Sequence, in Patients with Acquired Hemophilia Α

▪Acquired hemophilia A (AHA) is a rare autoimmune bleeding disorder, caused by auto-antibodies that inhibit the activity of endogenous human factor VIII (hFVIII). In most cases, the inhibition is governed by non-linear non-saturable (Type II) kinetics, resulting in incomplete inhibition and thus, underestimation of the FVIII inhibitor titer. A highly pure, B-domain deleted, antihemophilic FVIII (recombinant), porcine sequence (OBI-1) that is less susceptible to the inhibitory activity of anti-hFVIII antibodies has been developed as a replacement therapy to manage bleeding episodes in AHA patients.In one study of patients with AHA, the presence of pre-treatment anti-porcine FVIII (pFVIII) antibodies was reported not to correlate with the efficacy of a plasma-derived pFVIII1. However, cross-reactivity of anti-hFVIII antibodies with the recombinant pFVIII OBI-1 or de novodevelopment of anti-pFVIII antibodies could impair efficacy of OBI-1 treatment in subjects treated with pFVIII replacement therapy.This phase 2/3 open-label clinical trial investigated the efficacy and safety of OBI-1 in the treatment of serious bleeds in 28 adults with AHA. The primary efficacy endpoint was assessed at 24 hours (e.g., effective, partially effective). Subjects received an initial dose of OBI-1 (200 U/kg); additional doses were based on the subject’s target FVIII levels, anti-pFVIII titer, and clinical factors. This evaluation aims to address potential concerns regarding anti-pFVIII antibodies and their effect on the response to OBI-1 treatment in AHA patients.Overall 10/28 subjects (36%) had detectable anti-pFVIII inhibitors (range 0.8-29 BU) prior to treatment, presumably due to cross-reactive anti-hFVIII auto-antibodies also neutralizing pFVIII. The rise in FVIII activity level immediately after the first OBI-1 infusion was lower in subjects with baseline anti-pFVIII inhibitors ≥5 BU/mL (median rise: 29%, n = 4) than in subjects without detectable or with low anti-pFVIII inhibitor levels (<5 BU/mL, median rise: 96%; n=6). However, with tailored dosing all 10 subjects achieved clinically relevant FVIII activity levels. Furthermore, all had a positive response to treatment at or before 24 hours.All subjects received immunosuppressive therapy to treat their underlying autoimmune disease during the study. After treatment, 2 subjects with baseline anti-pFVIII inhibitors had an increase in anti-pFVIII titer (from 3 to 6 BU and from 15 to 183 BU); 8/10 subjects had a decline in anti-pFVIII titer to non-detectable levels. Five subjects did not have baseline anti-pFVIII but developed antibodies de novo (range 0.6-166 BU at final assessment). These de novo titers were first detected between 8 and 85 days after treatment initiation while the median duration of exposure to OBI-1 was 7 days. As 2 of these subjects had a concomitant increase in anti-hFVIII antibody titers, the de novoanti-pFVIII titers were possibly due to either, i) an anamnestic reaction to pFVIII that resulted in an increase in cross-reactive anti-hFVIII inhibitors, or ii) an immunogenic response to pFVIII yielding anti-pFVIII antibodies cross-reacting with hFVIII. Another 2/5 subjects had a concurrent decrease in anti-hFVIII antibody titers, suggesting an immunogenic response to pFVIII. These data indicate that immunosuppressive therapy used to treat auto-antibodies may not have the same suppressive effect on allo-antibodies.The presence of anti-pFVIII antibodies prior to treatment appears to have had an effect on the immediate FVIII activity level rise after the first dose, particularly if the titer was greater than 5 BU. However, this early low FVIII activity level could be overcome by a tailored dosing regimen, guided by measuring FVIII activity levels. Consistent with previous reports with a plasma-derived pFVIII1, our findings indicate a lack of correlation between baseline anti-pFVIII antibody titers and efficacy of OBI-1. OBI-1 replacement therapy facilitates objective and successful assessment of efficacy independently of inhibitors through monitoring of FVIII activity levels.1Morrison et al. (1993), Blood. 81:1513 DisclosuresFarin:Baxter Healthcare: Employment. Novack:Baxter Healthcare: Employment. Mo:Baxter Healthcare: Employment. Bourgeois:Baxter BioScience: Employment. Horling:Baxter BioScience: Employment. Ewenstein:Baxer Healthcare: Employment.

Does the presence of von Willebrand factor in FVIII-deficient plasma influences the measurement of FVIII inhibitor titres in haemophilia A patients?

Reliable measurement of FVIII inhibitor is critical in the follow-up of haemophilia A patients. We performed a multicentre study to evaluate whether the presence of von Willebrand factor (VWF) in FVIII-deficient plasma (FVIII-DP) influences FVIII inhibitor titres. Six French haematology laboratories participated in this study. Three samples with varying FVIII inhibitor titres (1, 5 and 15BU/mL) and one sample without any detectable FVIII inhibitor were tested using four different procedures for FVIII inhibitor assay. The Nijmegen method and a modified assay with imidazole were performed using FVIII-DP with and without VWF in the control mixture and as substrate plasma in the FVIII one stage assay (OSA). Each mixture (reference and test) was incubated for two hours at 37°C with buffered normal pool plasma. Higher inhibitor titres were measured in 5 and 15BU/mL samples when assays were performed with the Nijmegen method and FVIII-DP without VWF. When samples were diluted in imidazole buffer, similar inhibitor titres, close to expected values, were measured whether VWF was present in the FVIII-DP or not. The data obtained were also more accurate when residual FVIII activity levels between 40% and 60% were used to calculate inhibitor titres, despite a linear type I reaction kinetics. These results support the hypothesis that reliable FVIII inhibitor titres can be measured without the use of FVIII-DP containing VWF when an imidazole-modified assay is used.

Perioperative Management of Acquired Hemophilia A: A Case Report and Review of Literature

Introduction:Acquired hemophilia A is a rare bleeding disorder with a high mortality rate. Diagnosis and treatment of this disorder can be very challenging to anesthesiologists because of lack of a personal or familial abnormal bleeding history.Case presentation:We report a 60-year-old woman who presented to the operating room for an urgent fasciotomy. She was initially diagnosed to have compartment syndrome of her left upper extremity secondary to an expanding hematoma after multiple unsuccessful venipuncture attempts. After surgical intervention, she developed recurrent intramuscular hematomas, became severely anemic, and required surgical re-exploration and multiple blood product transfusions. Ultimately, she was found to have an elevated activated partial thromboplastin time (aPTT), very low FVIII activity, and high FVIII inhibitor titers consistent with the diagnosis of acquired hemophilia A.Conclusions:Treatment strategies in acquired hemophilia are based on two major objectives. During the acute stage, effective control of bleeding is critical. The ultimate therapeutic goal during the subacute phase is the elimination of the inhibitors targeting factor VIII. Here, we present this case and will review current literature regarding therapeutic approaches to this rare condition in the operating room setting and postoperative course.