Enzyme-linked immunosorbent assay (ELISA) of factor VIII antigen using a monoclonal antibody devoid of factor VIII inhibitor activity.

Abstract A circulating anticoagulant, which specifically inhibits Factor VIII (AHF), has been detected in some patients with hemophilia A who had received multiple transfusions. The inhibitor was quantitated by measurement of the degree of inactivation of Factor VIII. The data presented provide strong evidence for the antibody nature of the Factor VIII inhibitor in hemophilia: (1) All specific inhibitory activity of serum was detected in the γG globulin obtained by chromatography of the sera on DEAE cellulose. (2) Fab fragments obtained by digestion of the γG globulin with papain, contained 18-22 per cent of the specific inhibitory activity, while Fc fragments contained 0.4-3 per cent. F(ab')2 fragments obtained by digestion with pepsin contained 36-61 per cent of the specific inhibitory activity of γG globulin. (3) The level of the inhibitor of Factor VIII increased sharply following transfusions of blood and decreased slowly to its preinfusion level. (4) When small amounts of inhibitor were incubated in vitro with excess Factor VIII, the inhibitor activity was decreased. Infusion of Factor VIII into a patient with a low level of inhibitor decreased the inhibitor activity. (5) Clearance of the isologous inhibitor from the circulation of a normal subject was rapid.

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Quantitative Effects of the Reaction between Factor VIII and Factor VIII Inhibitors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653662 ◽

1971 ◽

Vol 26 (01) ◽

pp. 124-134

Author(s):

C. W McMillan ◽

R. C Elston

Keyword(s):

Factor Viii ◽

Factor Viii Inhibitor ◽

Inhibitor Activity ◽

Viii Inhibitor ◽

In Vitro Effects ◽

Logarithmic Relation

SummaryTo study the effects of factor VIII and factor VIII inhibitor on each other an assay was developed in which inhibitor activity could be directly related to units of factor VIII. Inhibitor activity in units per ml is defined as the number of factor VIII units reduced to 0.01 factor VIII unit by 1 ml inhibitor plasma after incubation for 1 h at 37° C. Whereas factor VIII was destroyed by inhibitor in accord with a double-logarithmic relation between initial and 1-h residual factor VIII activities, final inhibitor activity was found to be inversely proportional to the ratio of initial concentrations of factor VIII to inhibitor. The latter process resembles “dilution”, rather than direct neutralization, of inhibitor by factor VIII. These effects were observed both in the system for inhibitor assay and in more concentrated mixtures of factor VIII and inhibitor sources in vitro. Results of infusion of factor VIII into a subject with classic hemophilia and an acquired inhibitor suggested that in vivo and in vitro effects of factor VIII and inhibitor activities on each other are comparable, if not identical.

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Acquired Hemophilia Associated with Smoldering Myeloma: Demonstration That the Monoclonal Gammopathy Acts as the Factor VIII Inhibitor.

Blood ◽

10.1182/blood.v114.22.1307.1307 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1307-1307 ◽

Cited By ~ 1

Author(s):

Olivier Decaux ◽

Benoit Guillet ◽

Arnaud Millet ◽

Stephen Harding ◽

Arthur R. Bradwell ◽

...

Keyword(s):

Multiple Myeloma ◽

Factor Viii ◽

Binding Site ◽

Monoclonal Gammopathy ◽

Factor Viii Inhibitor ◽

Acquired Hemophilia ◽

Inhibitor Activity ◽

Acquired Haemophilia ◽

Viii Inhibitor ◽

Smoldering Myeloma

Abstract Abstract 1307 Poster Board I-329 A 44 years old woman presented with metrorrhagia. Coagulation tests revealed a prolongation of activated partial thromboplastin time up to 77 seconds (control 33 seconds) which could not be corrected by mixing with normal plasma. The factor VIII activity level was reduced to 0.06 IU/mL. The Bethesda assay demonstrated the presence of a factor VIII inhibitor at 29 Bethesda units per ml (BU/mL). Other coagulations studies were within the normal ranges. Serum protein electrophoresis revealed a monoclonal band in the gamma region. IgA kappa monoclonal gammopathy was confirmed by immunofixation. The concentration of IgA kappa was 2.1 g/L. The haemoglobin level was 13.1 g/dL, creatininemia 80 μmol/L and calcemia 2.38 mmol/L. Standard bone X Rays and MRI of spine showed no lytic bone lesions. Bone marrow aspiration showed 11% plasma cell infiltration. A diagnosis of IgA kappa smoldering multiple myeloma associated with acquired hemophilia was made. The patient initially received four Rituximab infusions without significant effect. Several episodes of moderate uterine bleedings were treated with recombinant activated Factor VII infusions. It is noteworthy that she never had any other bleeding. Finally, electrocoagulation of endometrium definitively stopped metrorrhagia despite persistence of factor VIII inhibitor activity. Given that the acquired haemophilia was no longer symptomatic and that there were no other complications associated with multiple myeloma no specific treatment of multiple myeloma was proposed. To investigate the link between the IgA kappa smoldering myeloma and the factor VIII inhibitor activity, polyclonal antibodies that are specific for IgA kappa or IgA lambda were used ex vivo. These antibodies target unique conformational epitopes located at the junctions of heavy chains and light chains of immunoglobulins (Binding Site Group Ltd). Bethesda assay was performed on patient's plasma diluted at 1/30 (patient's monoclonal IgA kappa final concentration: 0.07 g/L) mixed with either anti IgA kappa or anti IgA lambda antibodies (final concentration: 2.5 g/L). The anti factor VIII activity was decreased with addition of anti IgA kappa antibody (7.2 UB/ml) but not with anti IgA lambda antibodies (24 UB/ml). We confirmed these results by using a Polyethyleneglycol (PEG) precipitation step. Patient's plasma was mixed with anti IgA Kappa antibodies (final concentration 9.2 g/L) and PEG (3%). After centrifugation, the anti factor VIII inhibitor activity in supernatant was totally suppressed (< 0.4 UB/mL). As a control, the titre was unchanged (24 BU/mL) when anti IgA Lambda were used following the same protocol. These results suggest that the IgA monoclonal protein is responsible of factor VIII inhibitor activity. Acquired hemophilia is a rare autoimmune disorder in which the patients develop an autoantibody directed against coagulation factor VIII leading to a clinically significant bleeding diathesis. In 50% of the cases there is no detectable underlying disease; however in 10% of cases there is an association with an underlying malignancy, either solid or haematological. Concurrent acquired hemophilia and monoclonal gammopathy is rare and few reports have associated monoclonal proteins to factor VIII inhibition but without any in vitro confirmation. In this report, we described a case of an IgA kappa smoldering myeloma patient who presented with an acquired haemophilia and demonstrated that the IgA monoclonal protein was acting as the factor VIII inhibitor. A broad range of autoimmune phenomena frequently complicate hematologic malignancies and acquired hemophilia is considered to be the consequence of immune deregulation with usually oligoclonal anti-Factor VIII profile. Understanding of the underlying pathogenic mechanisms responsible for the acquired haemophilia could help to optimize treatment management. To the best of our knowledge, this is the first report of multiple myeloma associated acquired hemophilia with demonstration that monoclonal gammopathy could be responsible of factor VIII inhibitor activity. Disclosures Harding: The Binding Site Group Ltd: Employment. Bradwell:The Binding Site Group Ltd: Chairman.

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Transient factor VIII inhibitor and treatment with monoclonal-antibody-purified factor VIII

The Lancet ◽

10.1016/0140-6736(91)92891-5 ◽

1991 ◽

Vol 337 (8751) ◽

pp. 1222 ◽

Cited By ~ 1

Author(s):

JoséB. Montoro ◽

Santiago Rodríguez ◽

Carme Altisent ◽

JoanM. Tusell

Keyword(s):

Monoclonal Antibody ◽

Factor Viii ◽

Factor Viii Inhibitor ◽

Viii Inhibitor

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Immunoblot cross-reactivity of factor VIII inhibitors with porcine factor VIII

Blood ◽

10.1182/blood.v86.6.2183.bloodjournal8662183 ◽

1995 ◽

Vol 86 (6) ◽

pp. 2183-2190 ◽

Cited By ~ 13

Author(s):

K Koshihara ◽

J Qian ◽

P Lollar ◽

LW Hoyer

Keyword(s):

Factor Viii ◽

Human Factor ◽

Cross Reactivity ◽

The Other ◽

Enzyme Linked Immunosorbent Assay ◽

Factor Viii Inhibitor ◽

Human Factor Viii ◽

Viii Inhibitor ◽

A1 Domain ◽

Inhibitor Titer

Porcine factor VIII has been used successfully to treat factor VIII inhibitor patients whose plasmas have minimal cross-reactivity to porcine factor VIII. However, some inhibitor plasmas do inhibit porcine factor VIII, and the extent of procoagulant inhibition often increases after treatment with porcine factor VIII. Because there is no information about the porcine factor VIII epitopes with which these antibodies react, we have compared the immunoblot and enzyme-linked immunosorbent assay (ELISA) reactivities with porcine and human factor VIII for 20 inhibitor plasmas (11 from hemophilia A patients and 9 autoantibodies). Immunoblots identified binding to porcine factor VIII for only 2 of the 12 plasmas from patients who had not received porcine factor VIII, but this reactivity could not be predicted from the inhibitor titer to porcine factor VIII. Immunoblot reactivity with porcine factor VIII was detected for 7 of 8 inhibitor plasmas from patients who had been previously treated with porcine factor VIII, and the strength of this reactivity was generally related to the inhibitor titer. Of the 5 plasmas that were immunoblot positive with the porcine factor VIII A2 domain, 4 had inhibitor titers greater than 45 Bethesda units when tested with porcine factor VIII, whereas only 1 of 15 of the other plasmas had this level of inhibitor activity with porcine factor VIII. In contrast, immunoblot reactivity to the porcine factor VIII A1 domain did not correlate with the antiporcine VIII inhibitor titer. We also determined the effect of preincubation with human or porcine factor VIII on immunoblot reactivity. In one case, immunoblot reactivity with porcine factor VIII was absorbed with porcine, but not human, factor VIII, which is consistent with antibody formation after treatment with porcine factor VIII. In no cases did human factor VIII reduce the reactivity of inhibitor plasmas with the porcine A1 domain, suggesting that these antibodies are directed at unique porcine factor VIII determinants. The reactivity to porcine A2 in 2 plasmas probably represented cross-reactivity of similar A2 determinants, because it was absorbed by both human and porcine factor VIII. Although the ELISA assays with porcine factor VIII detected antibodies in some plasmas that could not be identified by inhibitor assay or immunoblot, the level of ELISA reactivity was generally consistent with the titers of the other assays.

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Epitope Specificity of Factor VIII Inhibitor Antibodies

Hämostaseologie ◽

10.1055/s-0038-1655342 ◽

1998 ◽

Vol 18 (03) ◽

pp. 121-128 ◽

Cited By ~ 1

Author(s):

Dorothea Scandella

Keyword(s):

Factor Viii ◽

Factor Viii Inhibitor ◽

C2 Domain ◽

Inhibitor Activity ◽

Epitope Specificity ◽

Factor Viii Concentrates ◽

Previously Treated Patients ◽

Previously Treated ◽

Viii Inhibitor ◽

Acidic Region

SummaryAntibodies that inactivate factor VIII develop most frequently in patients with severe hemophilia A, and they present a serious complication in the therapy of such individuals. Most patients with inhibitors have two or more different antibodies capable of factor VIII inactivation in their plasma, as shown by assays in which several factor VIII domain fragments are tested for neutralization of the inhibitor activity. Such neutralization assays of 23 patient plasmas suggest that there are three common epitopes. These epitopes have been localized by several methods to the A2, A3, and C2 factor VIII domains. Less common inhibitor epitopes lie within the heavy chain acidic region residues 336-372 and in a second region of the C2 domain. The light chain acidic region, residues 1649-1689, may also contain an inhibitor epitope, but this remains to be confirmed. The inhibitor epitopes of autoantibody patients are more restricted as half of them have anti-C2 domain antibodies that make up 95% of the inhibitor activity. The remainder have several inhibitors similar to those of the hemophiliacs. The predominant C2 domain epitope specificity was also seen in rare cases of inhibitor development due to two heat pasteurized factor VIII concentrates in previously treated patients without inhibitors. Inhibitors in mild hemophiliacs are rare as these individuals are usually tolerant to factor VIII. When tolerance is overcome in such patients, the immune responses characterized were diverse. In some patients there was a complete loss of tolerance.

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Acquired F VIII Inhibitor In An Identical Subhemophilic Twin

10.1055/s-0038-1652980 ◽

1981 ◽

Author(s):

J W ten Cate ◽

A Sturk ◽

C Breederveld

Keyword(s):

Complex Formation ◽

Factor Viii ◽

Genetic Predisposition ◽

Plasma Levels ◽

Factor Viii Inhibitor ◽

Inhibitor Activity ◽

Fviii Inhibitor ◽

Viii Inhibitor ◽

Inhibitor Titer ◽

High Responders

An identical (94,3% certainty) twin developed a factor VIII inhibitor, almost simultaneously, which may imply a genetic predisposition. Both patients were high responders (18 an 22 Bethesda U). The initial FVIII;C plasma levels gradually decreased from 0.24 U to 0.08 U. Upon transfusion of 2500 U human FVIII concentrate 75% was inactivated immediately and the remaining factor VIII;C activity had a T½ of 2 h. In order to investigate the T½ of their own FVIII, plasmaphoresis was performed in one of them and 500 ml plasma was cryo- precipitated as the first step for factor VIII purification. Surprisingly, only 10% of the FVIII;C present in plasma was recovered in the cryoprecipitate. FVIII was purified by Sepharose 6B chromatography. Inhibitor activity eluted in later fractions seperate from FVIII. The void volume fractions were pooled and were negative for FVIII inhibitor activity. Purified FVIII was radiolabeled (125I-lactoperoxidase technique) . CIg followed by autoradiogrphy and PAGE of the IFVIII, gave normal results. 12 u Cl 125I FVIII was injected i.v. and revealed a biphasic normal half life: T½ of the first phase was 2.0 h, of the 2nd phase 19.2 h. This may imply that this patient developed antibodies selectively directed against FVIII neoantigens present in the FVIII concentrates (T½ h:) . However, i.v. administration of DDAVP resulted in a decreasing FVIII inhibitor titer presumably due to complex formation of released endogenous FVIII the inhibitor. It is therefore concluded that 125I VIII formes complexes with the inhibitor and that these complexes are slowly cleared from the circulation.

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Acquired Haemophilia: Functional Study of Antibodies to Factor VIII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650189 ◽

1981 ◽

Vol 45 (03) ◽

pp. 285-289 ◽

Cited By ~ 14

Author(s):

J P Allain ◽

A Gaillandre ◽

D Frommel

Keyword(s):

Factor Viii ◽

Functional Study ◽

Factor Viii Inhibitor ◽

Acquired Haemophilia ◽

Viii Inhibitor ◽

Kinetics Of ◽

Antibody Function ◽

Native Plasma

SummaryFactor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR.Ag levels were found in all patients. VIII:WF levels were 50% of those of VTIIRrAg, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers.Antibody function has been characterized by kinetics of VIII :C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed:1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (> 1).2) Antibodies from other patients, usually with lower titres, inactivated VIII :C according to complex order kinetics, were not saturable, and had a less steep affinity slope (< 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

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A Higher than Expected Incidence of Factor VIII Inhibitors in Multitransfused Haemophilia A Patients Treated with an Intermediate Purity Pasteurized Factor VIII Concentrate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651565 ◽

1993 ◽

Vol 69 (02) ◽

pp. 115-118 ◽

Cited By ~ 131

Author(s):

Kathelijne Peerlinck ◽

Jef Arnout ◽

Jean Guy Gilles ◽

Jean-Marie Saint-Remy ◽

Jos Vermylen

Keyword(s):

Factor Viii ◽

Randomized Trials ◽

Specific Factor ◽

Factor Viii Inhibitor ◽

Haemophilia A ◽

Viral Inactivation ◽

Gradual Decline ◽

Inhibitor Level ◽

Factor Viii Concentrates ◽

Viii Inhibitor

SummaryIn May 1990, 218 patients with haemophilia A regularly attending the Leuven Haemophilia Center were randomly assigned to a group receiving either of two newly introduced factor VIII concentrates: factor VIII-P, an intermediate purity pasteurized concentrate, or factor VIII-SD, a high purity concentrate treated with solvent-detergent for viral inactivation.Patients were followed from May 1990 until October 1991. Between August 1991 and October 1991 a clinically important factor VIII inhibitor was detected in five out of the 109 patients receiving factor VIII-P while none of the 109 patients receiving factor VIII-SD developed such antibodies. All patients acquiring an inhibitor had previously been clinically tolerant to transfused factor VIII with 200 to more than 1,000 days of exposure to factor VIII prior to May 1990. Patients with inhibitors were transfused daily with 30 U factor VIII-SD per kg body weight, which was associated with a gradual decline of the inhibitor level. In all patients the antibodies were relatively slow-acting and predominantly directed towards the light chain of factor VIII.This study demonstrates a higher than expected incidence of factor VIII inhibitors associated with the use of a specific factor VIII concentrate in multitransfused haemophilia A patients. It indicates the usefulness of evaluating newly introduced concentrates in prospective, randomized trials.

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