A multicenter pharmacokinetic study of the B-domain deleted recombinant factor VIII concentrate using different assays and standards

Summary. When the one‐stage clotting assay is used in comparison with the chromogenic and immunological assays, plasma levels of factor (F)VIII are underestimated by 40–50% after infusion of B‐domain deleted recombinant FVIII (BDD‐rFVIII) in patients with hemophilia. A possible way to counteract the underestimation of FVIII levels by the one‐stage assay is the adoption of a recombinant FVIII reference standard instead of a plasma standard. To evaluate the usefulness of such a standard [ReFacto^® Laboratory Standard (RLS)], the pharmacokinetic parameters of a single dose of BDD‐rFVIII (25 U kg^?1) were evaluated in a multicenter study carried out in 18 patients with severe hemophilia A. The very low in vivo recovery, obtained with the combination of the one‐stage assay and plasma reference standard, was increased up to the values obtained by the chromogenic assay when the results were expressed in terms of RLS. When the plasma standard was used, the one‐stage/chromogenic ratio was 0.82 ± 0.12 for FVIII levels above 25 U dL^?1 and 1.42 ± 0.99 for FVIII levels below 25 U dL^?1. Using the RLS, the one‐stage/chromogenic ratio increased to 1.01 ± 0.19 at FVIII levels above 25 U dL^?1, as a consequence of a complete overlap of the two decays; however, at FVIII levels below 25 U dL^?1, the one‐stage/chromogenic ratio was still 1.6 ± 0.85. After the twelfth hour, FVIII concentrations obtained by chromogenic assay were always lower than those resulting from the one‐stage clotting assay, independently of the standard used. Results obtained by chromogenic assay were not affected by the type of standard used. Compared with those obtained by the one‐stage assay, higher values of clearance, lower volume of distribution area and shorter plasma half‐life or mean residence time were obtained by chromogenic assay because of a shape change of the decay curve due to a shift to higher values in the first part (time interval 0–12 h) and to lower values in the second part of the decay curve (time interval 12–48 h). As a consequence, the slope of the decay curve obtained by means of chromogenic assay was steeper. In conclusion, the more homogeneous results of in vivo recovery and pharmacokinetic analysis, due to the decrease of discrepancy between the two methods when RLS was used, make the cheaper and more widely used one‐stage assay preferable to the more expensive chromogenic assay, on condition that the ReFacto specific standard has used.     

Prevalence,biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one‐stage and chromogenic factor VIII activity

Summary. Background: In most laboratories, the severity of hemophilia A is assessed by the factor VIII activity (FVIII:C) one‐stage assay. However, comparisons of these results with those of two‐stage assays can reveal discrepancies and suggest misdiagnosis. Patients/Methods: In this monocentric study, we measured FVIII:C with two methods (one‐stage chronometric and chromogenic assays) in 307 (173 families) patients with moderate/mild hemophilia A. To compare results, we used a chronometric/chromogenic ratio. Discrepancy was defined as a ratio < 0.5 or > 1.5. We studied their putative involvement at known FVIII functional sites, their interspecies conservation status, and their spatial position within the FVIII structure. Results: Thirty‐six patients from 17 families exhibited a discrepancy between the two assays: 12 (6.9%) families had a low ratio (< 0.5), and five (2.9%) families had a high ratio (> 1.5). Qualitative deficiency was diagnosed in about 16% of the families. Molecular studies were performed in 15 of these 17 families, resulting in each case in the identification of missense mutations, including three novel mutations. We were further able to propose a pathophysiologic explanation. Conclusions: In this monocentric study, we have demonstrated a discrepancy between FVIII:C assay results in 10% of families with moderate/mild hemophilia A. The prevalence of ‘inverse’ discrepancy (i.e. low chronometric/chromogenic ratio) is high as compared with previous reports. We suggest that both FVIII:C assays are recommended in patients with moderate/mild hemophilia A for a complete biological phenotype. This could also improve our knowledge of the FVIII structure–function relationships.     

The influence of von Willebrand factor on factor VIII activity measurements

Summary.  Background: It has been reported by multiple laboratories that the quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure and the quality of reagents used in the assays. Objective: To evaluate the influence of von Willebrand factor (VWF) on FVIII activity in vitro . Methods: The activated partial thromboplastin time (APTT) and synthetic coagulation proteome assays were used. Citrated FVIII/VWF-depleted substrate plasma (SP) (both antigens < 0.5%) was used in all APTT assays. Results: The concentration of FVIII antigen in pooled plasma from healthy donors [normal plasma (NP)] was 1.5 n m . The SP reconstituted with 1.5 n m recombinant (r)FVIII clotted in 23.8 ± 0.2 s (standard deviation). The addition of 10 μg mL⁻¹ VWF to the SP increased the clotting time to 28.7 ± 0.1 s; that is, the activity of rFVIII (FVIIIc) decreased to 50%. This inhibitory effect of VWF decreased with decreasing rFVIII concentration in SP, and became negligible at rFVIII ≤ 10% (150 p m ). The FVIIIc of 1.5 n m FVIII in two products formulated with VWF (Immunate and Hemofil  M) was not affected by the addition of exogenous VWF to the SP, whereas the FVIIIc of the B-domainless rFVIII product ReFacto was decreased to 50% by the addition of VWF. In the synthetic coagulation proteome triggered with 5 p m tissue factor, VWF had no effect on thrombin generation. Conclusions: VWF has an inhibitory effect on the measurement of FVIII clotting activity. This effect depends upon the structure and formulation of the FVIII product.     

International comparative field study evaluating the assay performance of AFSTYLA in plasma samples at clinical hemostasis laboratories

Essentials

• AFSTYLA exhibits ≈50% underestimation in activity when the one‐stage (OS) assay is utilized.
• A field study compared the performance of AFSTYLA with Advate in factor VIII activity assays.
• AFSTYLA activity can be monitored with both the chromogenic substrate and the OS assay.
• The consistent OS underestimation allows for a conversion factor to be applied to OS results.

Summary

Introduction

AFSTYLA (antihemophilic factor [recombinant] single chain) is a novel B‐domain truncated recombinant factor VIII (rFVIII). For AFSTYLA, an approximate 50% discrepancy was observed between results of the one‐stage (OS) and chromogenic substrate (ChS) FVIII activity assays. An investigation was undertaken to test whether there is a linear relationship between ChS and OS assay results that would allow reliable clinical interpretation of results independent of the assay method used.

Aims

To provide confidence in future clinical monitoring, this field study investigated the performance of AFSTYLA and a full‐length rFVIII (Advate^®) in FVIII activity assays routinely performed in clinical laboratories.

Methods

The comparison of AFSTYLA and Advate was performed in an international, multicenter and blinded field study of simulated post‐infusion samples. The study documented the extent of variability between methods and laboratories and characterized the relationship between the ChS and OS assays.

Results

Results from 23 laboratories demonstrate that intra and interlaboratory variability in OS assays were similar for both products. When comparing within the OS assay format, there was a similar and reagent‐correlated variability in response to different activators for both AFSTYLA and Advate. The OS underestimation was highly predictable and consistent across the complete range of FVIII plasma concentrations.

Conclusion

Post‐infusion plasma AFSTYLA levels can be monitored in patients by the OS and ChS assays. The consistent and predictable difference between the two assay formats provides clinicians with adequate guidance on how to interpret the results of the OS assay using a single conversion factor.     

Accuracy of FVIII:C assay by one-stage method can be improved using hemophilic plasma as diluent

BACKGROUND: the basic prerequisite of Factor VIII clotting assay (FVIII:C) by One-Stage Method is that all other than FVIII clotting factors are present in constant concentration in each dilution of both standard reference and patient's plasma curves. On the contrary, the plasma content of each dilution is decreasing as the dilution factor increases. OBJECTIVES AND METHODS: to keep exactly constant the plasma content in each mixture, we performed all dilutions of both standard reference and patient's plasma with FVIII deficient plasma and further with a fixed amount of buffer (method B). To show the discrepancies between this method and regular method A, using buffer to make dilutions, a comparative study was conducted on FVIII: C assay on samples at known FVIII concentration and in patients' plasma. Imidazole or Owren's buffers and five different aPTT reagents were employed, both in method A and B. RESULTS: a discrepancy between FVIII: C assays obtained by method A and B was observed, mainly when Pathrontin SL and Imidazole buffer were used. The assays derived from method B always better fit with the expected, calculated, values of FVIII:C concentrations. Furthermore, FVIII: C was assayed in 60 patients: the outcome of method A was always higher than values of method B. The discrepancy between the two methods was higher at FVIII concentrations below 50 U/dL but null at 100 U/dL. The A slope was steeper than B slope and the difference was statistically significant starting from the 1/10 dilution. Accordingly, FVIII: C of patients' plasma obtained by method A was always higher that those obtained by method B, even 2 or 3 times for FVIII level < or = 10 U/dL or 1.4-1.6 times for FVIII levels between 10 and 25 U/dL. CONCLUSIONS: only method B is able to give FVIII: C assays in agreement with the expected values. The dilution of reference standards and samples with FVIII deficient plasma is crucial to accurately evaluate the post-infusion FVIII concentrations in pharmacokinetic studies or the trough level during prophylactic therapy and to investigate the discrepancy among different FVIII: C assays. In addition, the assessment of severity and classification of hemophilia should be reviewed.     

Genetic analysis of non-severe hemophilia A phenotype with A discrepancy between one-stage and chromogenic factor VIII activity assays

IntroductionThe diagnosis of hemophilia A (HA) is based on the measurement of factor VIII activity (VIII:C). About one-third of non-severe HA patients show a discrepancy of VIII:C measured by one-stage (VIII:C 1st) and chromogenic (VIII:C chr) assays. Different mutations in the F8 gene may cause the discrepancy in results of the FVIII activity assay.The aim of this study was to investigate F8 gene mutations in patients with assay discrepancies and to evaluate their impact on the results of VIII:C assays.MethodsMutation analysis was performed on 41 individuals with a discrepancy in VIII:C 1st and FVIII: C chr assays by direct sequencing. In addition, the effect of the variants on FVIII macromolecule structure was investigated by in silico and bioinformatics tools.ResultsGenetic analysis disclosed 22 different variants, of which 19 were identified for the first time to be involved in the phenotype of VIII:C discrepancy. Most of the variants related to the higher VIII:C 1st were found in A1, A2, A3 domains. The variant related to VIII:C chr > VIII:C 1st was located in the thrombin cleavage site. In silico analysis showed the effect of variants on FVIII macromolecule stability, which may be the possible mechanism causing the discrepancy.ConclusionOur data shed light on the impact of genetic defects on VIII:C assay and provided evidence that the consideration of these mutations may open a new window to the proper diagnosis and treatment monitoring of non-severe HA patients.     

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

Summary.  Introduction:  In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. Methods:  Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. Results:  The chromogenic FVIII:C levels were higher (0.90 ± 0.15 and 0.47 ± 0.13 IU mL⁻¹) than the 1-st clotting ones (0.14 ± 0.05 and 0.10 ± 0.05 IU mL⁻¹) in family X and Y, respectively ( P  < 0.001). Mean endogenous thrombin potential (ETP) was 1579 ± 359 n m  min⁻¹ and 1060 ± 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 n m  min⁻¹, whereas for those of family Y they ranged from 447 to 1122 n m  min⁻¹. Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. Conclusions:  Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.     

Thrombin generation assay using factor IXa as a trigger to quantify accurately factor VIII levels in haemophilia A

Summary. Background: The available methods for measuring factor VIII (FVIII) activity suffer reportedly from lack of sensitivity and precision in the < 1 IU dL^?1 range. This precludes correlation of clinical phenotype with FVIII levels. Objectives: To study a possible association between clinical phenotype in patients with FVIII levels < 1 IU dL^?1. Methods/Results: The FIXa‐driven FVIII assay (FVIII‐CAT) has a detection limit of 0.05 IU dL^?1. For the range of 0–2 IU dL^?1 FVIII, the intra‐assay coefficient of variation (CV) is around 2% and the inter‐assay CV is about 8%. We tested 30 hemophiliacs with FVIII:C between < 1 and 6 IU dL^?1 as measured in the one‐stage clotting assay using the FVIII‐CAT assay. For genetic defects related to moderate hemophilia, the FVIII‐CAT test finds FVIII levels that are in good agreement with those determined with the one‐stage assay. Of the 21 hemophilic patients with FVIII < 1 IU dL^?1, four patients exhibited a mild bleeding phenotype. When we applied TF‐initiated thrombin generation, patients with a mild clinical phenotype showed significantly higher endogenous thrombin potentials. Conclusion: The novel developed FVIII assay measures accurately FVIII levels below 1 IU dL^?1. Its application demonstrated that the clinical heterogeneity in individuals with < 1 IU dL^?1 FVIII is not associated with their FVIII level.     

Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein

Summary. Background: Hemophilia A results from a deficiency in factor VIII activity. Current treatment regimens require frequent dosing, owing to the short half‐life of FVIII. A recombinant FVIII–Fc fusion protein (rFVIIIFc) was molecularly engineered to increase the half‐life of FVIII, by 1.5–2‐fold, in several preclinical animal models and humans. Objective: To perform a biochemical and functional in vitro characterization of rFVIIIFc, with existing FVIII products as comparators.Methods: rFVIIIFc was examined by utilizing a series of structural and analytic assays, including mass spectrometry following lysyl endopeptidase or thrombin digestion. rFVIIIFc activity was determined in both one‐stage clotting (activated partial thromboplastin time) and chromogenic activity assays, in the context of the FXase complex with purified components, and in both in vitro and ex vivo rotational thromboelastometry (ROTEM) assays performed in whole blood. Results: rFVIIIFc contained the predicted primary structure and post‐translational modifications, with an FVIII moiety that was similar to other recombinant FVIII products. The von Willebrand factor‐binding and specific activity of rFVIIIFc were also found to be similar to those of other recombinant FVIII molecules. Both chromogenic and one‐stage assays of rFVIIIFc gave similar results. Ex vivo ROTEM studies demonstrated that circulating rFVIIIFc activity was prolonged in mice with hemophilia A in comparison with B‐domain‐deleted or full‐length FVIII. Clot parameters at early time points were similar to those for FVIII, whereas rFVIIIFc showed prolonged improvement of clot formation. Conclusions: rFVIIIFc maintains normal FVIII interactions with other proteins necessary for its activity, with prolonged in vivo activity, owing to fusion with the Fc region of IgG₁.     

Laboratory testing in hemophilia: Impact of factor and non‐factor replacement therapy on coagulation assays

The advent of extended half‐life (EHL) recombinant clotting factors and innovative non‐factor replacement therapeutics, such as emicizumab, offers several advantages over existing products for the prophylactic treatment of people living with hemophilia (PwH). These include low annual bleeding rates with less frequent dosing, higher trough plasma concentrations, and a more convenient route of administration. However, increasing use of these therapies poses challenges to clinicians and coagulation laboratories due to the lack of standardized assays for monitoring of hemostatic parameters, and the potential for misinterpretation of test results, which may jeopardize patient safety. Definitive diagnosis of hemophilia and treatment monitoring is reliant on demonstrating factor VIII (FVIII; hemophilia A) or factor IX (FIX; hemophilia B) deficiency using a functional coagulation assay. The most frequently used assays are based on activated partial thromboplastin time, using a one‐stage or two‐stage process. While one‐stage and chromogenic assays have performed well with human‐derived FVIII and FIX and full‐length recombinant products, EHL recombinant factors are heterogeneous in structure and mode of action and therefore show wide variation in activity levels between different one‐stage assays, and between one‐stage and chromogenic assays. In the context of the recommended stepwise approach for laboratory diagnosis of hemophilia, we examine the diagnostic challenges associated with the use of EHL factors and novel non‐factor therapeutics and consider the optimal diagnostic approach in PwH who are receiving these treatments. Ultimately, accurate diagnostic solutions are a prerequisite for personalized therapy to minimize treatment burden and improve quality of life in PwH.     

A collaborative study to establish the 7th International Standard for Factor VIII Concentrate.

A candidate concentrate, preparation N (99/678), was assayed and calibrated, as a potential replacement, against four established factor (F) VIII concentrate standards: the current WHO 6th International Standard (IS) (97/616), the previous 5th IS (88/640), the Mega 1 standard and Ph. Eur. BRP Batch 2 standard, in a collaborative study involving 38 laboratories. All laboratories were instructed to use the ISTH/SSC recommendations, including predilution of concentrates in FVIII-deficient plasma. Several laboratories performed more than one assay method and altogether there were 27 sets of assays with the one-stage method, 31 with the chromogenic method, and 18 with both methods. There was good agreement between laboratories using each of the two methods for comparison of preparation N against the four established standards, with overall potencies by one-stage and chromogenic methods differing only by less than 2%. However, there were significant differences in potencies relative to the different standards, ranging from 10.1 IU per ampoule against the Ph. Eur.BRP2 to 11.4 against the WHO 6th IS. Accelerated degradation studies showed that the proposed standard is very stable, with a predicted loss of activity per year of less than 0.001% at the recommended storage temperature of -20 degrees C. Various options for potency of preparation N were considered by the participants and by members of the ISTH/SSC FVIII/FIX Subcommittee. In November 2003, preparation N (NIBSC 99/678) was proposed to and accepted by the Expert Committee on Biological Standardization of the World Health Organization to be the 7th International Standard for Factor VIII Concentrate with an assigned potency of 11.0 IU per ampoule.     

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

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.

     

Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype

Severity of bleeding phenotype in hemophilia A (HA) depends on the underlying mutation in the F8 gene and, ultimately, on the concentration and functional integrity of the factor VIII (FVIII) protein in circulating plasma. Initial diagnosis for HA and monitoring of treatment is typically performed by measuring of FVIII activity by either one-stage assay or chromogenic assay. We review evidence for why both types of assay do not give comparable results in a significant proportion of patients with non-severe haemophilia A and why the discrepancy in results between both methods segregates with distinct subclasses of known missense mutations causing haemophilia A. The current understanding of the mechanistic basis for how FVIII:C assay discrepancies arise are discussed. Conclusion: We propose that both methods should be used in initial patient diagnosis along with follow-up genetic analysis to avoid potential misdiagnosis and to optimize treatment monitoring of patients with HA phenotypes.     

Effect of delayed blood processing on the yield of factor VIII in cryoprecipitate and factor VIII concentrate

Current standards for the preparation of factor VIII (FVIII) concentrates from human plasma recommend separation of plasma from red cells (RBCs) within 6 hours of blood donation, thereby reducing the volume of plasma from donated whole blood available for processing to FVIII concentrate. The decay of FVIII clotting activity (FVIII:C) in whole blood and plasma stored at 22 and 4 degrees C and the recovery of FVIII:C in cryoprecipitate and FVIII concentrate prepared from plasma separated from whole blood stored overnight at 4 degrees C were investigated. In whole blood stored at 22 degrees C and plasma stored at either 4 or 22 degrees C, 90 percent of the original FVIII:C was present at 6 hours, 80 percent at 12 hours, and 65 to 70 percent at 18 hours. At these times lower levels of FVIII:C were recovered from whole blood stored at 4 degrees C, that is, 84, 68, and 56 percent, respectively. In cryoprecipitates prepared from plasma separated from RBCs after 18 hours' storage at 4 degrees C (18-hour plasma), 43 percent of FVIII:C activity was recovered, as compared with 61 percent recovered from standard plasma separated within 6 hours of donation (6-hour plasma), p less than 0.05. With large-scale preparation of FVIII concentrates, however, the yield of FVIII:C was similar whether 18- or 6-hour plasma was used. Thus FVIII concentrates--but not cryoprecipitates--can be prepared from plasma separated from whole blood stored at 4 degrees C for up to 18 hours without undue loss of potency.     

The measurement of low levels of factor VIII or factor IX in hemophilia A and hemophilia B plasma by clot waveform analysis and thrombin generation assay

BACKGROUND: Precise assessment of clotting function is essential for monitoring of hemostatic treatment for hemophilias A and B. MATERIALS AND METHODS: Clot waveform analysis and thrombin generation assays were performed on factor (F) VIII- and FIX-deficient plasmas, which had been reconstituted with known amounts of recombinant FVIII (rFVIII) and affinity-purified FIX respectively. Clot waveforms were assessed qualitatively and quantitatively by measuring the parameters clotting time, maximum coagulation velocity (Min1), and maximum coagulation acceleration (Min2). The thrombin generation assay was also assessed qualitatively and measurements made of time to peak and peak height. RESULTS: Overall results obtained with both assays showed good correlation for both clotting factors confirming that the changes in clotting waveform reflected changes in thrombin generation. Both assays demonstrated a predictable dose response to the addition of FVIII or IX. However, clot waveform analysis was more sensitive than the thrombin generation assay, particularly in detecting very low levels (0-0.1 IU dL(-1)) of both factors. CONCLUSIONS: These data suggest that the application of clot waveform analysis to the routine management of the hemophiliacs could increase our understanding of the clinical significance of low levels of FVIII and FIX that cannot be measured by assays in current use. This may be particularly useful in the management of hemophiliacs with inhibitors or undergoing gene therapy.     

Induction of partial immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain

Summary. Background: The development of anti‐factor VIII (FVIII) neutralizing antibodies (inhibitors) is a significant obstacle to FVIII replacement therapy. Objective: As mucosal administration of an antigen may induce immune tolerance we have evaluated the efficacy of mucosal antigen exposure to achieve tolerance to FVIII. Methods: We investigated the effects of oral and nasal administration of the purified FVIII C2 domain (FVIII‐C2) to FVIII‐deficient BALB/c mice prior to FVIII protein challenge. Mice received oral or nasal doses of FVIII‐C2, followed by a subcutaneous challenge of either FVIII‐C2 or FVIII. The development of anti‐FVIII inhibitors, cytokine production by splenocytes in vitro, and adoptive transfer assays were analyzed. Results and Conclusions: Mucosal administration of FVIII‐C2 decreases the titer of anti‐FVIII‐C2 inhibitors after FVIII‐C2 challenge, and decreases the percentage of FVIII‐C2 specific antibodies after challenge with full‐length FVIII. Tolerance induction to FVIII‐C2 is associated with increased IL‐10 production by splenocytes in vitro, and can be adoptively transferred to naïve mice. This study is the first to demonstrate that tolerance to the FVIII‐C2 domain can be induced via the mucosal route. Based on these results, the potential use of FVIII‐specific mucosal tolerance induction as an immunotherapy treatment for anti‐FVIII inhibitor development warrants further investigation.     

Neutralization of antifactor VIII inhibitors by recombinant porcine factor VIII

BACKGROUND: Inhibitory antibodies to factor (F) VIII (FVIII inhibitors) present a major clinical challenge as a complication of hemophilia A and as acquired autoantibodies in non-hemophiliacs. Porcine FVIII is a potentially useful therapeutic agent because of its low crossreactivity with many inhibitors. Recombinant porcine FVIII (rpFVIII) is undergoing clinical trials in inhibitor patients. OBJECTIVES: The goals of this study were to neutralize human FVIII inhibitors in vitro with rpFVIII and to characterize the relationship between recovery of FVIII activity and the antiporcine FVIII inhibitor titer. METHODS: rpFVIII was added to 28 antihuman FVIII inhibitor plasmas and assayed either immediately or after a 2 h preincubation at 37 degrees C. The concentration of rpFVIII required for recovery of FVIII activity to 50% of normal (EC(50)) was determined by polynomial regression analysis and compared with the antiporcine FVIII inhibitor titer measured by Bethesda assay. RESULTS: Six of nine plasmas classified as non-crossreactive by Bethesda assay had detectable inhibitory activity in the FVIII recovery assay. Recovery was decreased following a 2 h preincubation compared with immediate assay for the 19 plasmas with detectable antiporcine FVIII inhibitors. There was a linear relationship between EC(50) and inhibitor titer for plasmas with antiporcine FVIII titers ranging from 0.6 to 10 BU per milliliter in both the immediate and the 2 h preincubation assays. CONCLUSION: A quantitative method for analysis of inhibitor neutralization in vitro has been developed, which may be useful for comparison with pharmacodynamic studies of rpFVIII in FVIII inhibitor patients and subsequent rational dosing in this patient population.     

Variability in factor VIII concentrate measurement: results from SSC field collaborative studies

Summary.  Seven 'field' collaborative studies on factor (F)VIII concentrate potency measurements were carried out, using local routine methodology, standards and calculation of results. Data from five of the 12 different concentrates studied are described in detail. These studies revealed that, for the intermediate-purity and the recombinant FVIII concentrates, one-stage potencies were significantly lower than chromogenic potencies, whilst for the two high-purity FVIII concentrates one-stage potencies were significantly greater than chromogenic potencies. On comparing predilution methods for the intermediate-purity concentrate, equivalent potencies were obtained using either buffer or FVIII-deficient plasma as prediluent. For the two high-purity and the recombinant concentrates, potencies obtained using buffer as prediluent were significantly greater and lower, respectively, than potencies obtained using FVIII-deficient plasma as prediluent. Interlaboratory variabilities were compared over all 12 concentrates studied and coefficients of variation (CVs) for one-stage assays were found to be much greater than for chromogenic assays. This was true for all concentrates except for the intermediate-purity concentrate and samples A and B from the first study, where the reverse was true. Furthermore, much better CVs were obtained when using FVIII-deficient plasma than when using buffer as prediluent, for all FVIII concentrates except for the intermediate-purity concentrate where the reverse was true, and sample B where CVs were equivalent. Overall, CVs were far worse than those obtained in controlled collaborative studies. Generally, however, CVs were better with chromogenic assays and predilution in FVIII-deficient plasma, as is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee, particularly for higher purity and recombinant concentrates.     

Factor VIII inhibitors: von Willebrand factor makes a difference in vitro and in vivo

Summary. Background: The important association between von Willebrand factor (VWF) and factor VIII (FVIII) has been investigated for decades, but the effect of VWF on the reactivity of FVIII inhibitory antibodies, referred to as inhibitors, is still controversial. Objective: To investigate the interaction among VWF, FVIII and FVIII inhibitory antibodies. Methods: Three sources of inhibitors were used for in vitro studies, including the plasma from immunized VWF^nullFVIII^null mice, purified plasma IgG from human inhibitor patients, or human monoclonal antibody from inhibitor patients’ B‐cell clones. Inhibitors were incubated with recombinant human FVIII (rhFVIII) either with or without VWF. The remaining FVIII activity was determined by chromogenic assay and inhibitor titers were determined. For in vivo studies, inhibitors and rhFVIII were infused into FVIII^null or VWF^nullFVIII^null mice followed by a tail clip survival test. Results: VWF has a dose‐dependent protective effect on FVIII, limiting inhibitor inactivation of FVIII in both mouse and human samples. A preformed complex of VWF with FVIII provides more effective protection from inhibitors than competitive binding of antibodies and VWF to FVIII. The protective effect of VWF against FVIII inactivation by inhibitors was further confirmed in vivo by infusing inhibitors and FVIII into FVIII^null or VWF^nullFVIII^null mice followed by a tail clip survival test. Conclusion: Our results demonstrate that VWF exerts a protective effect, reducing inhibitor inactivation of FVIII, both in vitro and in vivo.     

Determination of coagulation factor VIII activity by a chromogenic substrate method on STA, an automated coagulation analyzer

This study was aimed at evaluating the performance of a chromogenic factor VIII assay on STA, an automated coagulation analyzer. Additionally, a correlation study was conducted with an aPTT-based one-stage factor VIII clotting assay. Throughout the study the performance of the chromogenic assay was tested in two ranges of factor VIII activity: a high range with activity between 20% and 150% and a low range with activity below 20%. Inter-assay coefficient of variation ranged from 1.9% to 8.9% and intra-assay coefficient of variation from 0.5% to 11.4%, depending on factor VIII concentration. Day-to-day reproducibility was tested over a 5-day period; between-day imprecision ranged from 7.1% to 9.4%. The chromogenic factor VIII assay showed a good correlation with the clotting assay in both ranges. The correlation coefficients were 0.924 and 0.792 for the high and low range, respectively. A statistically significant difference in mean values was observed in the high range (p < 0.0001). Comparison between the chromogenic assay on STA versus on microplates showed a high correlation (0.991), which was highly significant (p < 0.0001). In conclusion, the chromogenic assay for factor VIII on STA shows good analytical performance. It correlates well with the one-stage factor VIII clotting assay, although significant differences between individual samples occur. Probably these are partly related to differences in measurement principle and standardization. Altogether, this precise and rapid assay is suitable for determination of factor VIII by an automated procedure.