Quantitation of Non-Inhibitory Anti-Factor VIII Antibodies in human Plasma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1025-1025
Author(s):  
Jolanta Krudysz-Amblo ◽  
Behnaz Parhami-Seren ◽  
Saulius Butenas ◽  
Kathleen E. Brummel-Ziedins ◽  
Edward D. Gomperts ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Hemophilia A ◽  
Positive Sample ◽  
Plasma Samples ◽  
Healthy Donors ◽  
Fviii Activity ◽  
Donor Population ◽  
Recombinant Fviii ◽  
Free Plasma

Abstract Hemophilia A is a clinically heterogeneous bleeding disorder characterized by the absence of functional factor VIII (FVIII). The most severe complication from exposure to exogenous factor VIII therapy is the development of alloantibodies that neutralize the effect of the therapeutic agent. The standard method for quantitation of FVIII inhibitors in human plasma is by the Nijmegen method, a modification of the Bethesda assay. Our objective was to develop a sensitive and specific fluorescence based immunoassay for the quantitation of anti-FVIII antibodies (FVIIIAb) in human plasma. An affinity purified human anti-FVIIIAb isolated from a hemophilia A patient was used as a calibrator, with a detectability limit of 40±1.5pM. The calibrator and the human plasma anti-FVIIIAb were captured on recombinant FVIII coupled microspheres and probed with mouse anti-human Ig conjugated to R-Phycoerythrin. Citrated human plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were studied and compared to 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1BU (94.6±0.08nM), 11BU (214.3±7.1nM), 106BU (2209.4±84.9nM) and 140BU (2417.7±3.8nM) as measured by the Nijmegen method. Among the 150 normal control plasmas, 4 subjects (3%) were determined as positive (anti-FVIIIAb ≥ 40pM) with concentrations in the range of 0.6nM to 1.9nM, well below that of 1BU (94.6±0.08nM) inhibitor-positive sample. Anti-FVIIIAb was also evaluated in seven commercial inhibitor-free plasma samples reported to contain <1% FVIII activity. Two of the seven plasmas were found to contain anti-FVIIIAb with concentrations of 112.9±1.9nM and 71.1±8.5nM. The concentration of the antibody in both samples was within the range of the measured molar concentration of an inhibitor-positive 1BU titer plasma sample. Thirty two inhibitor-negative hemophilia A subjects were also quantitated for anti-FVIIIAb. Eleven of the 32 samples tested positive for anti-FVIIIAb with molar concentrations in the range of 0.6–20nM. The overall prevalence of the non-inhibitory anti-FVIIIAbs in hemophilia A subjects evaluated by our immunoassay (n=39) is approximately 33% relative to healthy donor population in which 3% were determined as positive (≥40pM) for FVIIIAb.

scholarly journals Quantitation of anti–factor VIII antibodies in human plasma

Blood ◽  
2009 ◽  
Vol 113 (11) ◽  
pp. 2587-2594 ◽  
Author(s):  
Jolanta Krudysz-Amblo ◽  
Behnaz Parhami-Seren ◽  
Saulius Butenas ◽  
Kathleen E. Brummel-Ziedins ◽  
Edward D. Gomperts ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Hemophilia A ◽  
Plasma Samples ◽  
Healthy Individuals ◽  
Specific Fluorescence ◽  
Healthy Donors ◽  
Inhibitory Antibodies ◽  
Recombinant Fviii ◽  
20 Nm

The presence of antibodies (Abs) in hemophilia A patients can potentially influence the therapeutic qualities of factor VIII (fVIII) administration. Much work has been focused on the presence of inhibitory antibodies, whereas the quantitation of noninhibitory anti-fVIII antibodies has been largely undetermined. Our objective was to develop a sensitive and specific fluorescence-based immunoassay (FLI) for the quantitation of anti-fVIIIAbs in human plasma. Affinity-purified human anti-fVIIIAb, isolated from a hemophilia A subject, was used as a calibrator with a detectability limit of 40 (±1.5) pM. The calibrator and the human plasma anti-fVIIIAb were captured on recombinant fVIII (rfVIII)– coupled microspheres and probed with mouse anti–human Ig–R-phycoerythrin. Plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were compared with 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1 BU/mL (94.6 ± 0.8 nM), 11 BU/mL (214.3 ± 7.1 nM), 106 BU/mL (2209.4 ± 84.9 nM), 140 BU/mL (2417.7 ± 3.8 nM) as measured by the Nijmegen method. We also describe the validation of a mouse anti–human fVIIIAb as a surrogate calibrator. Four healthy individuals (3%) showed detectable anti-fVIIIAb in the range of 0.6 to 6.2 nM, whereas 13 (33%) of the 39 inhibitor-free hemophilia A subjects were positive for anti-fVIIIAb in the range of 0.5 to 20 nM. The method may be useful for therapeutic management of hemophilia A patients.

Development and Characterization of Recombinant Ovine Factor VIII

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1193-1193
Author(s):  
Philip M Zakas ◽  
Bagirath Gangadharan ◽  
Graca Almeida-Porada ◽  
Christopher D Porada ◽  
H. Trent Spencer ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Full Length ◽  
Light Chains ◽  
Fviii Activity ◽  
Sds Page ◽  
Bleeding Episodes ◽  
Activation Peptide ◽  
Recombinant Fviii

Abstract Abstract 1193 Factor VIII (fVIII) is an essential glycoprotein procofactor in the blood coagulation cascade. Mutations in the fVIII gene often result in diminished plasma fVIII activity causing the bleeding disorder hemophilia A, which obeys X-linked recessive genetics and affects approximately 1 in 7500 males. Current treatment is limited to intravenous infusion of plasma-derived or recombinant human (h)-fVIII containing products. This therapy is expensive and requires multi-weekly injections to achieve prophylaxis, which must be maintained for the duration of the patients' life to avoid debilitating joint disease as well as life-threatening bleeding episodes. While gene therapy is being explored as a potential cure, much research is aimed at improving the therapeutic utility of recombinant fVIII. Our laboratory has been interested in the comparison of recombinant fVIII molecules derived from different animal species and has identified many species-specific biochemical properties. The characterization of recombinant murine (m)-factor VIII revealed near complete stability at physiologic concentrations following thrombin activation upon which h-fVIII activity decays on the order of minutes. In contrast, porcine (p)-fVIII demonstrates 100-fold increased post-translational biosynthesis over h-fVIII as well as decreased engagement of the endoplasmic reticulum-resident unfolded protein response. Arruda and colleagues showed that canine (c)-fVIII displays 3-fold higher specific activity than that of h-, p-, or m-fVIII and currently is being used to treat bleeding episodes in canine hemophilia A colonies. Finally, we have generated hybrid fVIII constructs, mapped the sequences necessary and sufficient for certain differential properties, identified immunogenic epitopes inherent to each species and created novel fVIII molecules with combined potentially beneficial characteristics. The current study is a continuation of this line of research focusing on ovine (o)-fVIII. Recently, a line of hemophilia A sheep was re-established and the pathology and clinical profile was described. O-fVIII possesses a high degree of homology to h-fVIII with a similar A1-A2-activation peptide-A3-C1-C2 domain structure. The causative mutation was identified as a single nucleotide insertion causing a frameshift and premature stop codon in exon 14. Administration of human and hybrid h/p-fVIII in these sheep corrected the bleeding phenotype transiently, but invariably induced the formation of high titer anti-fVIII inhibitory antibodies leading to premature mortality. Herein, we describe the generation, expression and biochemical characterization of recombinant full-length and B-domain-deleted (BDD) o-fVIII. O-fVIII was cloned into the mammalian expression vector, ReNeo and a pace/furin linker sequence was introduced between the A2 domain and the activation peptide. Utilizing a baby hamster kidney cell expression system, full-length o- and h-fVIII demonstrated similar expression levels at 0.098 ± 0.024 and 0.022 ± 0.006 units/106 cells/24hr, respectively (p = 0.99). Removal of the ovine B domain resulted in increased expression (0.97 ± 0.2 units/106 cells/24hr, p < 0.001) to a level equivalent to BDD h-fVIII (0.49 ± 0.149 units/106 cells/24hr; p = 0.06). BDD o-fVIII was purified to virtual homogeneity from conditioned serum-free media using a two-step ion-exchange chromatography procedure identified previously for the purification of recombinant h- and p-fVIII. Two independent preparations were analyzed and determined to have specific activities of 12,300 and 14,760 units/mg. SDS-PAGE analysis revealed three predominant polypeptides species, with a minority of intact single chain as well as predominantly processed heavy and light chains. Uniquely, the heavy and light chain polypeptides displayed similar relative mobility and could only be distinguished by thrombin proteolysis or immuno-precipitation prior to SDS-PAGE using monoclonal antibodies with known epitopes in either the heavy or light chains. We anticipate that recombinant o-fVIII, similar to the situation that has occurred with the canine hemophilia A colony and recombinant c-fVIII, will facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for research and development of novel hemophilia A therapeutics. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Congenital hemophilia A with low activity of factor XII: a case report and literature review

2021 ◽  
Vol 47 (1) ◽  
Author(s):  
Baoyu Lei ◽  
Chuang Liang ◽  
Haiyan Feng
Keyword(s):  
Genetic Testing ◽  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Coagulation Factor Viii ◽  
Factor Xii ◽  
Fviii Activity ◽  
Recombinant Fviii ◽  
Low Activity

Abstract Background Congenital hemophilia A is a recessive inherited hemorrhagic disorder. According to the activity of functional coagulation factors, the severity of hemophilia A is divided into three levels: mild, moderate and severe. The first bleeding episode in severe and moderate congenital hemophilia A occurs mostly in early childhood and mainly involves soft tissue and joint bleeds. At present, there are limited reports on severe congenital hemophilia A with low factor XII (FXII) activity during the neonatal period. Case presentation A 13-day-old neonate was admitted to the hospital with hematoma near the joints of both upper arms. Coagulation tests showed he had low activity of factor VIII (FVIII) and FXII. He was diagnosed with congenital hemophilia A and treated with human coagulation factor VIII (recombinant FVIII). Although the hematoma became smaller, FVIII activity was only increased to a certain extent and FXII activity decreased gradually. Unfortunately, the child responded poorly to recombinant human coagulation factor VIII and his guardian rejected prophylactic inhibitors and genetic testing and refused further treatment. Three months later, the child developed intracranial hemorrhage (ICH) due to low FVIII activity. Conclusions In hemophilia A, the presence of FVIII inhibitors, drug concentration and testing are three important aspects that must be considered when FVIII activity does not reach the desired level. Early positive disease treatment and prophylaxis can decrease the frequency of bleeding and improve quality of life. We recommend that pregnant women with a family history of hemophilia A undergo early prenatal and neonatal genetic testing.

Genetics and Hemostatic Potential in Persons with Mild to Moderate Hemophilia A with a Discrepancy between One-Stage and Chromogenic FVIII Assays

2020 ◽  
Author(s):  
Annelie Strålfors ◽  
Danijela Mikovic ◽  
David Schmidt ◽  
Liselotte Onelöv ◽  
Nida Mahmoud Hourani Soutari ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Genetic Alterations ◽  
Plasma Samples ◽  
Background Factor ◽  
Novel Mutations ◽  
Endogenous Thrombin Potential ◽  
One Stage ◽  
Fviii Activity ◽  
A Domains

Abstract Background Factor VIII (FVIII) activity (FVIII:C) can be measured by different methods including one-stage clotting assays (OSAs) and chromogenic assays (CSAs). Discrepancy between FVIII:C assays is known and associated with genetic variations causing mild and moderate hemophilia A (HA). We aimed to study the discrepancy phenomenon and to identify associated genetic alterations. Further, we investigated if hemostatic global assays could discriminate the group with discrepant FVIII:C from them. Methods The study contained plasma samples from 45 patients with HA (PwHA) from Hemophilia Centers in Stockholm, Sweden, and Belgrade, Serbia. We measured FVIII:C with OSA and CSA, sequenced the F8 gene, and performed two global hemostatic assays; endogenous thrombin potential and overall hemostatic potential. Results Nineteen of 45 PwHA had a more than twofold higher FVIII:C using OSA compared to CSA and were considered discrepant. Thirty-four causal mutations were detected, where of five had not previously been associated with assay discrepancy. These novel mutations were p.Tyr25Cys, p.Phe698Leu, p.Met699Leu, p.Ile1698Thr, and Ala2070Val. We found no difference between discrepant and nondiscrepant cases with either of the global assays. Conclusion There was a discrepancy between FVIII:C assays in almost half of the PwHA, which for some could lead to missed HA diagnoses or misclassification of severity. Genotyping confirmed that mutations associated with FVIII:C discrepancy cluster in the A domains of F8, and five mutations not previously associated with FVIII:C discrepancy was identified. Global hemostatic assays did not contribute to distinguish assay discrepancy in PwHA.

scholarly journals Activity of transgene-produced B-domain–deleted factor VIII in human plasma following AAV5 gene therapy

Blood ◽  
2020 ◽  
Vol 136 (22) ◽  
pp. 2524-2534 ◽  
Author(s):  
Steffen Rosen ◽  
Stefan Tiefenbacher ◽  
Mary Robinson ◽  
Mei Huang ◽  
Jaydeep Srimani ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Factor Xa ◽  
Activity Levels ◽  
Adeno Associated Virus ◽  
Gene Therapies ◽  
Molecular Features ◽  
Fviii Activity ◽  
Recombinant Fviii

Abstract Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically use a B-domain–deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) assays than in chromogenic-substrate (CS) assays, whereas recombinant FVIII-SQ products had lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (international units per milligram) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assay kits and clinical laboratories, suggesting that intrinsic molecular features are potential root causes. Further experiments in 2 participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from nonhemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate end point to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on clinicaltrials.gov as #NCT02576795 and #NCT03370913 and, respectively, on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database; https://eudract.ema.europa.eu) as #2014-003880-38 and #2017-003215-19.

scholarly journals Detection of Hemophilia a Carriers by Repeated Factor VIII Activity/Antigen Determinations

1977 ◽  
Author(s):  
U. Seligsohn ◽  
A. Zivelin ◽  
H. Peretz ◽  
M. Modan
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Bayes Theorem ◽  
Plasma Samples ◽  
Factor Viii Activity ◽  
Relative Odds ◽  
Specificity And Sensitivity ◽  
Fviii Activity ◽  
Fresh Plasma ◽  
The Mean

Detection of hemophilia A carriers (C), using factor VIII (fVIII) activity (Ac) and antigenicity (Ag) measurements, has been hampered by an overlap of results obtained in normal females (NF) and obligatory carriers (OC). In an attempt to improve the specificity and sensitivity of C detection, 29 OC and 33 NF were examined 3 times. FVIII Ac and fVIII Ag were determined in fresh plasma samples, employing a one stage method and the Laurell technic with rabbit anti fVIII antiserum, respectively. Using the mean of the 3 pairs of measurements, a discriminant function, based on Bayes’ theorem, was obtained for calculating the probability that a woman is a C. Three ranges of measurements were defined: Definite C, possible C and definite NF. The results were as follows:If only the first pair of measurements was used, the results were much less satisfactory:Thus, for each suspected C, the relative odds of being a C can be assigned by using this function, but the results should be based on the mean of 3 measurements.

Investigations on the Nature of Hemophilia A

1963 ◽  
Vol 09 (01) ◽  
pp. 030-052 ◽  
Author(s):  
Eberhard Mammen
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Barium Sulfate ◽  
Freeze Drying ◽  
Room Temperature ◽  
Hemophilia A ◽  
Normal Human Plasma ◽  
Normal Human ◽  
And Storage ◽  
Prothrombin Activation

SummaryIn this paper an inhibitor is described that is found in hemophilic plasma and serum different from any till now described inhibitor. The inhibitor only inhibits prothrombin activation in the “intrinsic clotting systems”. This inhibitor is probably not present in normal human plasma or serum. It is destroyed by ether and freeze drying, is labile to acid and storage at room temperature. It is stable upon dialysis and has not been adsorbed on barium sulfate, aluminum hydroxide or kaolin. It precipitates at 50% v/v saturation with alcohol. The nature of this inhibitor seems to be a protein or lipoprotein.Factor VIII was isolated from hemophilic plasma. The amount isolated was the same as from normal plasma and the activity properties were not different. Hemophiliacs have normal amounts of factor VIII.

A Multicenter Pharmacosurveillance Study for the Evaluation of the Efficacy and Safety of Recombinant Factor VIII in the Treatment of Patients with Hemophilia A

1997 ◽  
Vol 78 (05) ◽  
pp. 1352-1356 ◽  
Author(s):  
Emel Aygören-Pürsün ◽  
Inge Scharrer ◽  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Parvovirus B19 ◽  
Subjective Evaluation ◽  
Recombinant Factor Viii ◽  
Fviii Inhibitor ◽  
Previously Treated Patients ◽  
Previously Treated ◽  
Bleeding Episodes ◽  
Recombinant Fviii

SummaryIn this open multicenter study the safety and efficacy of recombinant factor VIII (rFVIII) was assessed in 39 previously treated patients with hemophilia A (factor VIII basal activity ≤15%).Recombinant FVIII was administered for prophylaxis and treatment of bleeding episodes and for surgical procedures. A total of 3679 infusions of rFVIII were given. Efficacy of rFVIII as assessed by subjective evaluation of response to infusion and mean annual consumption of rFVIII was comparable to that of plasma derived FVIII concentrates. The incremental recovery of FVIII (2.4 ± 0,83%/IU/kg, 2.12 ± 0.61%/IU/kg, resp.) was within the expected range. No clinical significant FVIII inhibitor was detected in this trial. Five of 16 susceptible patients showed a seroconversion for parvovirus B19. However, the results are ambiguous in two cases and might be explained otherwise in one further case. Thus, in two patients a reliable seroconversion for parvovirus B19 was observed.

Development of neutralizing antibodies in application of various concentrates of blood coagulation factor VIII in the treatment of hemophilia A

2021 ◽  
Author(s):  
Н.И. Зозуля
Keyword(s):  
Factor Viii ◽  
Cell Line ◽  
Neutralizing Antibodies ◽  
Hemophilia A ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Human Cell Line ◽  
Chinese Hamster Cell ◽  
Coagulation Factor Viii ◽  
Recombinant Fviii

Серьезным осложнением, связанным с лечением гемофилии А, является развитие ингибиторов. В последние годы был проведён ряд исследований, посвящённых данной проблеме: RODIN, INSIGHT, FranceCoag, SIPPET и NuProtect. В данном обзоре суммируются основные результаты этих исследований. Согласно результатам рандомизированного исследования SIPPET, препараты плазматического фактора свертывания крови VIII (FVIII) обладают меньшей иммуногенностью, чем препараты рекомбинантного FVIII, синтезированного из клеточной линии китайских хомячков, что следует учитывать при выборе стратегии лечения. Согласно результатам исследования NuProtect, опубликованным в 2019 г., концентрат рекомбинантного FVIII, полученный из клеточной линии человека, демонстрирует профиль иммуногенности, сходный с таковым у препаратов плазматического FVIII. У ранее нелеченых пациентов с ненулевыми мутациями при применении симоктоког альфа не наблюдалось образования ингибиторов, также как и в случае применения препаратов плазматического FVIII в исследовании SIPPET. Inhibitor development is a serious complication associated with hemophilia A therapy. A number of studies have been carried out of this issue — RODIN, INSIGHT, FranceCoag, SIPPET, and NuProtect. This review summarizes the main results of these studies. According to the results of the SIPPET randomized trial, plasma-derived coagulation factor VIII (FVIII) products are less immunogenic than recombinant FVIII products synthesized from a Chinese hamster cell line; this fact should be taken into account in choosing a treatment strategy. According to the results of NuProtect study published in 2019, the concentrate of human cell line-derived recombinant FVIII demonstrates immunogenicity profi le similar to the one in plasma-derived FVIII products. Previously untreated patients with non-zero mutations receiving simoctocog alfa did not show development of inhibitors as well as in case of administration of plasma-derived FVIII products in SIPPET study.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

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