Nijmegen-Bethesda Assay Research Articles

Evaluation of nonneutralizing antibodies against factor VIII in severe haemophilia A patients from India.

: Haemophilia A is treated by replacement therapy with factor VIII (FVIII) concentrate. This strategy of treatment is ineffective in some patients due to the development of neutralizing antibodies (NNAs) against FVIII. The inhibitors have been identified to act against the functional domains of FVIII. The presence of NNAs against FVIII has also been identified. There is limited data on the prevalence and significance of NNA in haemophilia. To identify the presence of NNA in severe haemophilia A in our population, patients who were recruited from community-based camps were evaluated for FVIII activity. The patient's samples were further analysed for inhibitor activity with Nijmegen-Bethesda Assay and for NNAs using an in-house ELISA. 312 severe haemophilia patients were analysed for inhibitors and NNA. In-house ELISA picked up antibodies in 56 patients (17.9%). Of these 42 (13.7%) had inhibitory antibodies and in 14 patients (4.5%) there was no evidence of FVIII inhibitory activity. A substantial number of patients with severe haemophilia A have NNA. Continuous long-term follow-up is required in this cohort to evaluate the significance of this observation.

Comparative analysis of chromogenic vs clot based cdc modified, nijmegen-bethesda assay for detection of factor VIII inhibitor titre

Background:-Inhibitors to infused factor VIII are the most significant complication of hemophilia treatment. These inhibitors are usually IgG antibodies, that react with FVIII in a time and temperature dependent manner. Coagulation factor VIII inhibitors can be detected by Chromogenic, clot based and immunological assays. However, there is lack of consensus as to what constitutes a positive inhibitor, including the appropriate cut-off for inhibitor measurement The main objective of this study is to compare the sensitivity and specificity of chromogenic Nijmegen Bethesda assay (CNBA) with Centre for disease control modified Nijmegen Bethesda (CDC-NBA) assay against the Reference control method (RCM).Materials and Methods: The Coagulometer used for inhibitor titre quantification is Sysmex CS-5100. APTT reagent used isPathromtin SL supplied by seimensSeimens. All data were expressed as Mean ± SD. Statistical formulae were used for sensitivity and specificity calculations. Unpaired students t test was used whereever necessary and a P value of 2 NBU) CNBA has 88% and 80% and CDC-NBA has 85 and 70 % sensitivity and specificity respectively.Conclusion :- These results shows that CNBA is more sensitive and specific than CDC-NBA at both low and high inhibitor titre. Moreover chromogenic assays can differentiate factor specific inhibitor from nonspecific inhibitors like lupus anticoagulant and unfractionated heparin therapy.Keywords: Hemophilia, Bethesda assay, ELISA, Factor VIII, Inhibitor, Mixing studyAbbrevations: APLA- Antiphospholipid antibody syndrome, CDC:NBA- Centers for Disease Control and Prevention - Nijmegen-BethesdaAssay, CNBA:- chromogenic Nijmegen Bethesda assay

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.

Optimization of pre-analytical heat treatment for inhibitor detection in haemophilia A.

Factor VIII (FVIII) antibody formation is the greatest clinical and laboratory challenge within the haemophilia centre. The Nijmegen-Bethesda assay (NBA) is the gold standard for inhibitor quantification, but affected by pre-analytical variables including a patient's FVIII activity (FVIII:C). Pre-analytical heat treatment (PHT) provides a methodology for inhibitor testing when measurable FVIII:C is present. We evaluated the effect of different PHT conditions (time/temperature) on FVIII:C as well as on potency of inhibitory activity in samples containing FVIII:C (endogenous pooled plasma and exogenous recombinant FVIII (rFL-FVIII) concentrate) or FVIII inhibitor. PHT of endogenous FVIII at 37°C, 47°C and 52°C resulted in declining measurable FVIII:C at 120minutes (69%, 57% and 13% of the original FVIII:C, respectively). Incubation at 56°C resulted in FVIII:C≤1IU/dL after 60minutes for endogenous FVIII and 120minutes for rFL-FVIII. Incubation at 58°C resulted in FVIII:C<1IU/dL at 15-30minutes for endogenous FVIII and at 30-60minutes for rFL-FVIII. No difference was seen for inhibitor detection following PHT (56°C or 58°C) by NBA or anti-FVIII IgG ELISA. PHT at 58°C for 30minutes demonstrated consistent reduction in FVIII:C<1IU/dL without appearing to affect inhibitor detection. Laboratory awareness of differences in thermostability of different sources of FVIII is important when choosing PHT conditions.

Effects of pre-analytical heat treatment in factor VIII (FVIII) inhibitor assays on FVIII antibody levels.

The use of pre-analytical heat treatment (PHT) with the Nijmegen-Bethesda assay (NBA) for inhibitors to factor VIII (FVIII) can remove/destroy infused or endogenous FVIII from patient plasma samples, allowing testing of recently infused patients with haemophilia. Two PHT methods have been described as follows: heating to 56°C for 30minutes and heating to 58°C for 90minutes. Data examining the effects of PHT on anti-FVIII IgG4 , the antibodies known to correlate most closely with the presence of FVIII inhibitors, are limited. To assess the effect of PHT on the levels of detectable anti-FVIII IgG4 . Nijmegen-Bethesda assay-positive specimens were incubated at 56, 58 or 60°C for 90minutes, and anti-FVIII IgG4 was measured by fluorescence immunoassay (FLI) at 30-minute intervals. The effects of PHT on the ability of recombinant FVIII (rFVIII) to inhibit detection of patient antibodies by FLI was also examined to assess the stability of rFVIII under the various PHT conditions tested. Levels of anti-FVIII IgG4 showed little change following incubations at 56°C (mean 101% of original value at 30minutes and 100% at 60minutes) but decreased upon exposure to 58°C (mean 85% at 30minutes and 66% at 60minutes). In addition, heating to 56°C effectively decreased the ability of rFVIII to block antibody binding compared to unheated rFVIII. The optimal temperature for PHT in the FVIII NBA is 56°C. Higher temperatures may lead to loss of inhibitory antibodies.

Limit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors

Background The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, with the use of 'more sensitive methods' for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen-Bethesda assay [NBA]) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with sensitive immunologic methods. Objective To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytic heat inactivation to liberate bound anti-FVIII antibodies. Methods Specimens with known inhibitors were tested with the CDC-NBA. IgG4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). Results Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). With four statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG4 anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥ 0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU. Conclusions The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG4 in some patients with negative (< 0.5) NBU. The sharp increase in IgG4 frequency between 0.4 and 0.5 NBU validates the established threshold for positivity of ≥ 0.5 NBU for the CDC-NBA, supporting the need for method-specific thresholds.

Feiba Global Outcome Study (FEIBA-GO): Long-Term Real World Data on Apcc (FeibaR) in Patients with Inhibitors. First Demographic Data

Feiba Global Outcome Study (FEIBA-GO): Long-Term Real World Data on Apcc (FeibaR) in Patients with Inhibitors. First Demographic Data

Survey of the anti–factor IX immunoglobulin profiles in patients with hemophilia B using a fluorescence‐based immunoassay

Background Hemophilia B (HB) is an inherited bleeding disorder caused by the absence or dysfunction of coagulation factor IX (FIX). A subset of patients who have HB develop neutralizing alloantibodies (inhibitors) against FIX after infusion therapy. HB prevalence and the proportion of patients who develop inhibitors are much lower than those for hemophilia A (HA), which makes studies of inhibitors in patients with HB challenging due to the limited availability of samples. As a result, there is a knowledge gap regarding HB inhibitors. Objective Evaluate the largest group of patients with inhibitor-positive HB studied to date to assess the relationship between anti-FIX antibody profiles and inhibitor formation. Methods A fluorescence immunoassay was used to detect anti-FIX antibodies in plasma samples from 37 patients with HB. Results Assessments of antibody profiles showed that anti-FIX IgG1-4 , IgA, and IgE were detected significantly more often in patients with a positive Nijmegen-Bethesda assay (NBA). All NBA-positive samples were positive for IgG4 . Anti-FIX IgG4 demonstrated a strong correlation with the NBA, while correlations were significant, yet more moderate, for anti-FIX IgG1-2 and IgA. Conclusions The anti-FIX antibody profile in HB patients who develop inhibitors is diverse and correlates well with the NBA across immunoglobulin (sub)class, and anti-FIX IgG4 is particularly relevant to functional inhibition. The anti-FIX fluorescence immunoassay may serve as a useful tool to confirm the presence of antibodies in patients who have low positive NBA results and to more clearly define, predict, and treat alloantibody formation against FIX.

A variation of the Nijmegen-Bethesda assay using heat or a novel heat/cold pretreatment for the detection of FIX inhibitors in the presence of residual FIX activity.

The inclusion of a heat treatment step has improved the classic Nijmegen-Bethesda assay for detection of factor VIII (FVIII) inhibitors in the presence of residual FVIII activity (FVIII:C). However, information regarding heat-modified Nijmegen-Bethesda assays for the detection of FIX inhibitors is still limited. Three methods to measure FIX inhibitors in the presence or absence of residual FIX activity (FIX:C) using three different activated partial thromboplastin time (aPTT) reagents were investigated. These included the standard Nijmegen-Bethesda assay (method 1), a heat-modified assay (method 2) and a novel heat/cold-modified assay (method 3). In the absence of FIX:C, all methods measured similar levels of FIX inhibitor, while FIX inhibitor titres varied widely in the presence of residual FIX:C. Using method 1, inhibitors were not accurately measured in the presence of residual circulating FIX:C. Using method 2, detection was improved, especially at higher inhibitor titres. Using method 3, some additional sensitivity was obtained. The choice of aPTT reagent did not affect the detection of inhibitors. Heat pretreatment is recommended for detecting FIX inhibitors in samples with residual FIX:C. The heat/cold modification improved the sensitivity of the Nijmegen-Bethesda assay, resulting in higher tolerance for residual FIX:C.

Diagnostic and prognostic value of factor VIII binding antibodies in acquired hemophilia A: data from the GTH‐AH 01/2010 study

Essentials Factor VIII (FVIII) binding IgG detected by ELISA could be an alternative to the Bethesda assay. We studied the performance of anti-FVIII IgG ELISA in patients with acquired hemophilia and controls. Anti-FVIII IgG > 99th percentile of controls was highly sensitive and specific. Patients with high anti-FVIII IgG have a lower chance of achieving remission. Background Acquired hemophilia A is a severe bleeding disorder that requires fast and accurate diagnosis as it occurs often unexpectedly in previously healthy men and women of every age. The Nijmegen-modified Bethesda assay is the diagnostic reference standard for detecting neutralizing autoantibodies against factor VIII (FVIII), but is not widely available, not ideal for quantifying the complex type 2 inhibitors seen in acquired hemophilia, and suffers from high inter-laboratory variability. Objectives To assess the diagnostic and prognostic value of FVIII-binding antibodies as detected by ELISA compared with the Nijmegen Bethesda assay. Methods Samples from the time of first diagnosis and clinical data were available from 102 patients with acquired hemophilia enrolled in the prospective GTH-AH 01/2010 study. Controls (n = 102) were matched for gender and age. Diagnostic cut-offs were determined by receiver-operator curve analysis. The prognostic value was assessed in 92 of the 102 patients by Cox regression analysis of time to partial remission. Results Anti-FVIII IgG above the 99th percentile (> 15 arbitrary units per mL) revealed high sensitivity and specificity (both 0.99; 95% confidence interval, 0.95-1.0) for diagnosing acquired hemophilia. The likelihood of achieving partial remission was related to anti-FVIII IgG concentration (< 300 arbitrary units, 1.0; 300-1050, 0.65; > 1050, 0.39). The Bethesda titer was only associated with the likelihood of partial remission when analyzed in the central laboratory, but not when data from local GTH study sites were used. Conclusion Although the Nijmegen-modified Bethesda assay is the reference standard for demonstrating neutralizing antibodies, the detection of FVIII-binding antibodies by ELISA is similarly sensitive and specific for diagnosing acquired hemophilia. In addition, anti-FVIII IgG may provide prognostic information.

An Unusual Case of Acquired Hemophilia a and Factor XIII Consumption

BACKGROUND: Acquired hemophilia A (AHA) results from auto-antibodies that neutralize factor VIII (FVIII) coagulant function. AHA is rare, usually occurring late in life, and occasionally post-partum. It is most often idiopathic, but can be associated with malignancy, autoimmunity, or drugs. In contrast to congenital hemophilia, AHA generally manifests as mucocutaneous bleeding, with poor correlation to antibody titer or residual FVIII activity. Management goals are the achievement of hemostasis and antibody eradication. Here we describe an unusual case of AHA and concomitant factor XIII consumption in a young nulliparous woman.CASE REPORT: A 24-year-old woman with a symptomatic congenital choledochal cyst underwent a Roux-en-Y bypass, bile duct resection and cholecystectomy. Abdominal sepsis ensued from extended spectrum beta-lactamase Klebsiella, with ertapenem initiation on post-operative day (POD) 5. On POD6 she developed severe intra-abdominal bleeding associated with prolongation of the activated partial thromboplastin time (aPTT) from 35 seconds (pre-operative) to 40 seconds (reference interval 28-35 seconds). On POD10, FVIII activity and FVIII inhibitor titer, using the modified Nijmegen Bethesda assay, were measured at 0.08 U/ml and 4.5 BU/ml, respectively. In addition to supportive transfusion therapy, the patient received a sequence of desmopressin (0.3 mcg/kg IV), recombinant FVIII (50 U/kg), and activated prothrombin complex concentrate (aPCC) (50-75 U/kg IV q8h) with tranexamic acid (10-20 mg/kg IV q8h) from POD13-20. Prednisone was concomitantly initiated (1.5 mg/kg/day) (Figure 1). Bleeding persisted despite reduction in clotting time on rotational thromboelastometry post-aPCC. As neither intensified aPCC nor recombinant factor VIIa (rFVIIa) (80 mcg/kg IV q2h) provided substantial benefit, recombinant porcine factor VIII (rpFVIII) was administered from POD20-28 (100 U/kg IV qd). RpFVIII resulted in hemostatic improvement until anti-porcine FVIII antibodies developed on POD27. On POD28, she was transferred to the local hemophilia treatment center where complete factor analysis identified concurrent FXIII deficiency [0.08 U/ml] using a latex immunoassay. FXIII inhibitor analysis using a chromogenic assay was negative. The peak FVIII inhibitor titer and nadir FVIII activity were 74 BU/ml and <0.01 U/ml, respectively. APCC was resumed [POD28-37] and plasma derived factor XIII (pdFXIII) was given every 4 days to maintain FXIII >0.20 U/ml. The patient was also treated with rituximab [POD28,36,43,50]. On POD37, massive intra-abdominal bleeding prompted sequential aPCC, rFVIIa and pd von Willebrand factor (VWF):FVIII concentrate. Once the FVIII inhibitor titer began to wane, bypassing therapy was discontinued and she was managed solely with pdVWF:FVIII. On POD47 all FVIII-related support was discontinued, as the FVIII inhibitor was undetectable and endogenous FVIII normalized. Her FXIII consumption and requirement for FXIII replacement, however, persisted.DISCUSSION: The unusual features of this case of AHA include the patient's young age, the possible triggers for the FVIII inhibitor (extensive abdominal surgery, sepsis, and/or carbapenem use), and concomitant FXIII deficiency. We hypothesize that FXIII deficiency was secondary to consumption from the massive abdominal wound given its persistence despite normalization of hepatic function. Repeated life-threatening bleeding required trials of multiple hemostatic agents. Persistent and prolonged bleeding despite ongoing FVIII replacement should trigger investigation for a concomitant hemostatic deficiency. AHA is a rare condition, and treatment requires intensive monitoring, clinical experience and specialized laboratory support. Current guidelines are largely derived from expert opinion, and contemporary case descriptions for rare disorders are essential to advance options in best care. [Display omitted] DisclosuresSt. Louis:Baxter/Baxalta: Consultancy. Sholzberg:CSL Behring: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding; Amgen: Honoraria.

Heat treatment of samples improve the performance of the Nijmegen–Bethesda assay in hemophilia A patients undergoing immune tolerance induction

Nijmegen–Bethesda assay is the gold standard to assess inhibitory antibodies against factor (F) VIII. This method has some limitations, including high coefficient of variation and possible interference of residual endogenous or exogenous factor VIII. Heat-treatment of samples at 56°C for 30min could be a strategy to improve the sensitivity of this test. The aim of this study was to compare inhibitor quantification in hemophilia patients with and without inhibitor performed in previously heated and non-heated samples. A total of 109 analyses from 46 patients with severe hemophilia A were performed. Patients were divided into three groups: 20 patients with no history of inhibitor, recently and not recently exposed to FVIII (group I), 21 patients with history of inhibitor not exposed to FVIII (group II), and 5 patients (68 samples) undergoing an immune tolerance induction (ITI) protocol (group III). For patients with no history of inhibitor, heat-treatment did not modify the results (p=0.24). However, differences in inhibitor levels between heated and non-heated samples were observed in patients with history of inhibitor (group II, p<0.05) and in patients in ITI (group III, p<0.001). In 11 samples, inhibitor quantification shifted from negative to positive. Additionally, a longitudinal evaluation of each ITI patient showed similar trend line for the results of heated and non-heated samples. In this study, we demonstrated that heating samples increase sensitivity of Nijmegen–Bethesda assay, with no shift from negative to positive results in patients with no history of inhibitor. Furthermore, this procedure has an important role to patients undergoing an ITI protocol.

A longitudinal evaluation of anti-FVIII antibodies demonstrated IgG4 subclass is mainly correlated with high-titre inhibitor in haemophilia A patients.

The development of inhibitory antibodies against factor VIII (FVIII) (inhibitor) is the major complication in haemophilia A patients. The FVIII-binding antibodies development comprises a polyclonal immunoglobulin (Ig) G response. Recent studies showed strong correlation between the presence of neutralizing anti-FVIII antibodies (inhibitors) and IgG4 subclass. The aim of this study was to evaluate anti-FVIII IgG subclasses in haemophilia A patients with inhibitor both in a cross-sectional and in a longitudinal analysis. Inhibitors were determined by Nijmegen-Bethesda assay. Anti-FVIII IgG subclasses were performed by ELISA, and samples from 20 healthy individuals were used to validate the test. We studied 25 haemophilia A patients with inhibitor, previously treated exclusively with plasma-derived FVIII concentrates or bypassing agents. The IgG subclasses distributions were evaluated in two groups of patients classified according to inhibitor response. IgG1 and IgG4 antibodies were most prominent in haemophilia A patients with inhibitors when compared with IgG2 and IgG3. This study reports for the first time the behaviour of FVIII-binding IgG1 and IgG4 subclasses in a longitudinal analysis, in a clinical setting, of high-response inhibitor haemophilia A patients, showing the correlation of IgG4 and the inhibitor titres. In spite of being considered a non-pathologic antibody subclass with anti-inflammatory properties in other situations, IgG4 is correlated with the presence of high-titre inhibitor in the haemophilia setting. The comprehension of the IgG4 role in immune response may be crucial to establish the process for designing specific tolerance to FVIII.

Characterization of the anti‐factor VIII immunoglobulin profile in patients with hemophilia A by use of a fluorescence‐based immunoassay

The development of neutralizing antibodies, referred to as inhibitors, against factorVIII is a major complication associated with FVIII infusion therapy for the treatment of hemophiliaA (HA). Previous studies have shown that a subset of HA patients and a low percentage of healthy individuals harbor non-neutralizing anti-FVIII antibodies that do not elicit the clinical manifestations associated with inhibitor development. To assess HA patients' anti-FVIII antibody profiles as potential predictors of clinical outcomes. A fluorescence immunoassay (FLI) was used to detect anti-FVIII antibodies in 491 samples from 371 HA patients. Assessments of antibody profiles showed that the presence of anti-FVIII IgG1 , IgG2 or IgG4 correlated qualitatively and quantitatively with the presence of an FVIII inhibitor as determined with the Nijmegen-Bethesda assay (NBA). Forty-eight patients with a negative inhibitor history contributed serial samples to the study, including seven patients who had negative NBA titers initially and later converted to being NBA-positive. The FLI detected anti-FVIII IgG1 in five of those seven patients prior to their conversion to NBA-positive. Five of 15 serial-sample patients who had a negative inhibitor history and had anti-FVIII IgG1 later developed an inhibitor, as compared with two of 33 patients with a negative inhibitor history without anti-FVIII IgG1 . These data provide a rationale for future studies designed both to monitor the dynamics of anti-FVIII antibody profiles in HA patients as a potential predictor of future inhibitor development and to assess the value of the anti-FVIII FLI as a supplement to traditional inhibitor testing.

A Case Series Evaluating the Use of a Von Willebrand Factor/Factor VIII Concentrate (Wilate®) for Immune Tolerance Induction (ITI) in Children with Severe Factor VIII Deficiency

Introduction: Development of circulating anti-factor VIII antibodies (inhbitors) is the most serious and challenging complication in the treatment of hemophilia A. Up to 38% of hemophilia patients develop inhibitors with recombinant FVIII (rFVIII) products (Gouw et al. N Engl J Med. 2013; 368:231-239). The presence of inhibitors leads to an increased risk of bleeding, poor physical functioning and quality of life (Benson et al., Eur. J. Haematol. 2012; 88:371-379). Immune tolerance induction (ITI) is the most common method for eliminating inhibitors, historically performed with high dose, and prolonged treatment with plasma-derived (pd), or recombinant FVIII (rFVIII) concentrates. Although ITI for the eradication of inhibitors has become standard of care for hemophilia patients the therapeutic superiority of a particular product type (rFVIII vs. pd-FVIII) has not yet been conclusively demonstrated. In accordance with its role in stabilizing FVIII, the presence of von Willebrand factor (VWF) in pd-FVIII concentrates has been shown to improve the outcome of ITI. Wilate® (Octapharma) is a high-purity human plasma derived complex containing two proteins (VWF and FVIII) in a 1:1 ratio. The aim of this study was to determine the effectiveness of Wilate for primary ITI therapy for six patients with severe hemophilia A.Patients and Methods: The case history for six pediatric hemophilia A patients prior to and during primary Wilate ITI was reviewed. For 5/6 patients, inhibitors developed during rFVIII factor replacement therapy. For the sixth patient, inhibitors were detected at the time of hemophilia diagnosis. ITI began once patients achieved an inhibitor titer of less than 10 BU/mL. The ITI dosing regimen ranged from 50-60 IU/Kg of Wilate three times per week to 200 IU/Kg once daily. Inhibitor titers were measured regularly, prior to and during ITI using the Nijmegen-Bethesda assay. The number of port-a-cath infections and bleeding episodes were also monitored. ITI success was defined as: an undetectable inhibitor level (<0.6 BU/mL), FVIII plasma recovery ≥ 66% of predicted, and FVIII half-life ≥6 hours.Results: Wilate ITI was well tolerated in all patients, with no product-related adverse events. All patients had a port-a-cath device inserted for Wilate injections. Two port-a-cath infections occurred during ITI. Five of six patients had poor prognostic factors for ITI outcome. These poor prognostic factors included a high-risk FVIII gene mutation, historical peak inhibitor titer greater than 50 BU/mL, age of ITI onset greater than 6 years, and ITI onset more than 12 months from inhibitor development. The frequency of these poor prognostic factors varied amongst the patients: 1 patient had 4, 1 patient had 2, and 3 patients presented with 1 poor prognostic factor. Despite the presence of these high-risk factors, Wilate was successful at reducing the inhibitor titers to undetectable levels in all patients. Furthermore, inhibitor titers have remained low or undetectable without significant spikes for the duration of treatment. Patient plasma recovery and FVIII half-life results have also indicated that patients are progressing towards successful ITI. Importantly, for 6/6 patients (including 3 patients who had previously been treated with Anti-Inhibitor Coagulant Complex (FEIBA) prophylaxis therapy) - Wilate therapy was successful at reducing the number of bleeding episodes allowing for the cessation of FEIBA prophylaxis. Since commencing Wilate ITI, 6/6 patients have not reported any major bleeding episodes. The improved clinical outcome was perceived by the patients as an improved well-being, and quality of life.Conclusion: Wilate ITI was found to be well tolerated, safe, and successful at reducing inhibitor levels to below the detectable range for six severe hemophilia A patients. Patients experienced no treatment related adverse events, had a low rate of port-a-cath infections, and did not present with any major bleeding episodes while on Wilate ITI. In light of the 3-5 fold increase in overall treatment costs of immune tolerance induction, careful consideration should be given to choice of product (rFVIII versus pd-FVIII) – especially for patients at high-risk of failure. (Dimichele et al. Haemophilia 2004: 10 Suppl 4;140-145). The present data suggest that Wilate, a pd-FVIII product, is effective in managing patients with inhibitors. DisclosuresBelletrutti:Baxter Canada: Honoraria, Membership on an entity’s Board of Directors or advisory committees; CSL Behring Canada: Honoraria.

Efficacy, Immunogenicity, Pharmacokinetics, and Safety Of Human-cl rhFVIII – a GCP Study In Children With Severe Haemophilia A

IntroductionHuman-cl rhFVIIIis the first recombinant factor VIII concentrate expressed in a human cell line (Human Embryonic Kidney 293F cells). Studies in adolescent and adult pre-treated patients with severe haemophilia A showed a favorable PK profile with a median half-life of 17.1 hours (one stage assay) and indicated safety and efficacy in preventing and treating bleeding episodes (BE). AimsThe objectives of this international GCP study were to evaluate the efficacy, safety, immunogenicity and pharmacokinetic (PK) properties of Human-cl rhFVIIIin previously treated children between 2 and 12 years of age. MethodsAll patients started with an in-vivo recovery (IVR) investigation and in a subset of patients, the PK of Human-cl rhFVIII was assessed in comparison to the patient's previously used FVIII product. After an injection of 50 IU/kg, blood samples were collected up to 48 hours for PK analysis and up to 2 hours for IVR. IVR was repeated in all patients after 3 and 6 months. FVIII plasma levels were measured by chromogenic and one-stage assay in a central laboratory. Patients were treated prophylactically with Human-cl rhFVIII every other day or 3x weekly, injecting 30-40 IU Human-cl rhFVIIIper kg for at least 6 months and > 50 exposure days. Inhibitors were measured before, during and at the end of the study by modified Nijmegen Bethesda assay in a central laboratory. Adverse events were recorded throughout the study. Results59 patients (29: 2-5 years; 30: 6-12 years) were enrolled from 15 specialized sites in Europe. 13 children of each age group participated in the comparative PK investigation. Mean PK parameters of Human-cl rhFVIII were similar in both age groups for both the chromogenic and the one-stage assay: AUCnorm 0.23 vs. 0.24 h*IU/mL/[IU/kg]); IVR 1.88 vs. 1.61% per IU/kg; T1/2 9.7 vs. 12.5 h. IVR remained stable throughout the study. A total of 108 BEs in 32/59 patients were treated with Human-cl rhFVIII. The majority of treated BEs were traumatic (60.2%) and minor (56.5%). The mean ± SD monthly bleeding rate of all types of BEs/patient was 0.34±0.43 (spontaneous BEs: 0.12±0.27; traumatic BEs: 0.19±0.29). No inhibitor development was reported; no patient discontinued the study because of an AE. ConclusionThe data indicate that Human-cl rhFVIII is efficacious and safe in preventing and treating BEs in previously treated children. The PK of Human-cl rhFVIII was comparable in both studied age groups. Disclosures:Liesner:Octapharma AG: Consultancy, Investigator Other. Jansen:Octapharma AG: Employment. Klukowska:Octapharma AG: Investigator Other. Vdovin:Octapharma AG: Investigator Other. Szczepanski:Octapharma AG: Investigator Other. Knaub:Octapharma AG: Employment.

Validation of 4% albumin as a diluent in the Bethesda Assay for FVIII inhibitors

External quality assurance programs show the Nijmegen Bethesda Assay for FVIII inhibitors improves test specificity compared to the Classic Bethesda Assay but its uptake has been slow possibly due to the cost of using FVIII deficient plasma as diluent. This study was conducted to determine if modifying the Nijmegen Bethesda assay by replacement of FVIII deficient plasma with 4% as a diluent would be suitable for for measuring FVIII inhibitors. The titres of 59 samples from 35 patients with FVIII inhibitors were determined in parallel tests by the Nijmegen Bethesda Assay and and the modified Nijmegen assay. Method reproducibility was assessed on inhibitor-containing samples from seven individuals covering a range of titres from 1-200 Bethesda units/mL. The all-sample geometric mean titre was 6.73 Bethesda units/mL for the Nijmegen Bethesda Assay and 7.54 Bethesda units/mL for the modified Nijmegen assay. No sample was found where a difference in measured titre between methods would have altered clinical management. Agreement was very close in samples with titres less than 2BU/mL. Both assays gave inhibitor titres in external quality assurance samples of close to consensus values. The average between-run coefficients of variation were 8.6% for the Nijmegen Bethesda Assay and 7.9% for the modified Nijmegen assay. The modified Nijmegen assay using 4% albumin as the sample diluent showed good overall comparability to our existing Nijmegen Bethesda Assay and is substantially more cost-effective, making it a reasonable alternative for measuring FVIII inhibitors.

Diagnosis and treatment of factor VIII and IX inhibitors in congenital haemophilia: (4th edition)

Peter W. Collins, Elizabeth Chalmers, Daniel P. Hart, Ri Liesner, Savita Rangarajan, Kate Talks, Mike Williams and Charles R. Hay School of Medicine, Cardiff University, University Hospital of Wales, Wales, Royal Hospital for Sick Children, Glasgow, The London School of Medicine and Dentistry, Royal London Hospital, Barts, Queen Mary University, London, Great Ormond Street NHS Trust, London, Hampshire Hospital NHS Foundation Trust, Basingstoke & North Hampshire Hospital, Basingstoke, Royal Victoria Infirmary, Newcastle upon Tyne, Birmingham Childrens’ Hospital NHS Foundation Trust, Birmingham and Central Manchester University Hospitals, Manchester, UK

Validation of Nijmegen–Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance

As part of a pilot U.S. inhibitor surveillance project initiated at the Centers for Disease Control and Prevention (CDC) in 2006, a centralized inhibitor measurement was instituted. To validate a modified method for inhibitor measurement suitable for surveillance of treated and untreated patients. In all, 710 subjects with hemophilia A were enrolled; 122 had a history of inhibitor (HI). Nijmegen-Bethesda assay (NBA) results on 50 split specimens shipped on cold packs and frozen were equivalent (r=0.998). Because 55% of 228 initial specimens had factor (F)VIII activity (VIII:C) present, a heat treatment step was added. Heating specimens to 56°C for 30 min and centrifuging removed FVIII, as demonstrated by a reduction of VIII:C and FVIII antigen to <1 U dL(-1) in recently treated patients. Among specimens inhibitor-negative before heating, one of 159 with negative HI and five of 30 with positive HI rose to ≥ 0.5 Nijmegen-Bethesda units (NBU) after heating. Correlation of heated and unheated inhibitor-positive specimens was 0.94 (P=0.0001). The modified method had a coefficient of variation (CV) for a 1 NBU positive control of 10.3% and for the negative control of 9.8%. Based on results on 710 enrollment specimens, a positive CDC inhibitor was defined as ≥ 0.5 NBU. Results were similar when 643 post-enrollment specimens were included. Of 160 enrolled hemophilia B patients, two had HI. All others had NBU ≤ 0.2 at enrollment. The CDC experience demonstrates that this modified NBA can be standardized to be within acceptable limits for clinical tests and can be used for national surveillance.