Bethesda Assay Research Articles

Low factor VIII recovery in haemophilia A patients without inhibitor titre is not due to the presence of anti‐factor VIII antibodies undetectable by the Bethesda assay

Measurements of factor VIII (FVIII) recovery in previously untreated patients with haemophilia A were done as part of the clinical trial of safety and efficacy of the recombinant FVIII, Recombinate™. In 22 of 72 assessable patients, positive inhibitor titres ≥ 0.6 Bethesda units mL−1 were detected by the Bethesda assay in one or more plasma samples, and the remaining 50 patients were negative at all timepoints. Of the latter group, 16 individuals without inhibitors unexpectedly had both normal (111) and low (52) recoveries during the study. We investigated the possibility that other antibodies not detectable in the Bethesda assay were responsible for the low recovery, by using a highly sensitive immunopreciptation (IP) assay for detection of all antiFVIII antibodies. Eight of the 16 patients with low and normal recoveries did indeed have antibodies detected by the IP assay, and the remaining eight were negative. Four antibody‐positive individuals had insignificantly low titres, and the other four had modest to high titres. In the latter group, antibodies were found with similar frequencies and titre in plasmas from patients with low or normal recovery. Low recovery in haemophilia A patients without inhibitor titres must therefore be attributed to factors other than antiFVIII.

Low factor VIII recovery in haemophilia A patients without inhibitor titre is not due to the presence of anti-factor VIII antibodies undetectable by the Bethesda assay.

Measurements of factor VIII (FVIII) recovery in previously untreated patients with haemophilia A were done as part of the clinical trial of safety and efficacy of the recombinant FVIII, Recombinate. In 22 of 72 assessable patients, positive inhibitor titres > or = 0.6 Bethesda units mL-1 were detected by the Bethesda assay in one or more plasma samples, and the remaining 50 patients were negative at all timepoints. Of the latter group, 16 individuals without inhibitors unexpectedly had both normal (111) and low (52) recoveries during the study. We investigated the possibility that other antibodies not detectable in the Bethesda assay were responsible for the low recovery, by using a highly sensitive immunopreciptation (IP) assay for detection of all antiFVIII antibodies. Eight of the 16 patients with low and normal recoveries did indeed have antibodies detected by the IP assay, and the remaining eight were negative. Four antibody-positive individuals had insignificantly low titres, and the other four had modest to high titres. In the latter group, antibodies were found with similar frequencies and titre in plasmas from patients with low or normal recovery. Low recovery in haemophilia A patients without inhibitor titres must therefore be attributed to factors other than antiFVIII.

Antigenicity of putative phospholipid membrane-binding residues in factor VIII

AbstractMost inhibitory antibodies to human factor VIII (fVIII) bind to epitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of binding of fVIII to phospholipid. The x-ray structure of the human fVIII C2 domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, and Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part of a ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively charged phosphatidylserine. In this study, 8 active mutants of human fVIII (Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu, Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, and Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on the basis of differences between human, porcine, murine, and canine fVIII at proposed phospholipid binding sites, were expressed. The antigenicity of the mutants toward 5 C2-specific polyclonal human antibodies was measured by using the Bethesda assay. A human monoclonal anti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody, NMC VIII-5, were also included in the analysis. In comparison with wild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252 single mutants had lower antigenicity toward most of the inhibitors. In contrast, the Val2223Ala and Lys2227Glu mutants usually showed increased antigenicity. These results suggest that C2 inhibitors frequently target the Met2199/Phe2200 and Leu2251/Leu2252 β-hairpins and are consistent with the hypothesis that these residues participate in binding to phospholipid membranes. In contrast, Val2223 and Lys2227 may oppose antibody binding sterically or through stabilization of a low-affinity membrane-binding conformation of the C2 domain.

Blockade of CD40/CD40 Ligand Interactions Prevents Induction of Factor VIII Inhibitors in Hemophilic Mice but Does not Induce Lasting Immune Tolerance

Patients with severe hemophilia A frequently develop neutralizing anti-factor VIII antibodies after replacement therapy with factor VIII (FVIII). In a search for new strategies to induce immune tolerance against FVIII in these patients, we used a murine model of hemophilia A to investigate the importance of CD40/CD40 ligand (CD40L) interactions for the initiation of the anti-FVIII immune response. We focused our attention in particular on the induction of neutralizing anti-FVIII antibodies and the Th1/Th2 polarization of FVIII-specific T cells. The development of anti-FVIII antibodies was analyzed by ELISA systems (detection of total anti-FVIII antibodies) and Bethesda assays (determination of neutralizing anti-FVIII antibodies). Factor VIII-specific T cells were characterized by multiparameter flow cytometry and cytokine ELISAs for the detection of cytokine production in splenic CD4+ T cells after in vitro restimulation with FVIII. Hemophilic mice received four doses of FVIII and anti-CD40L antibody MR1 (24 h before FVIII). Subsequently mice received four doses of FVIII only. The induction of neutralizing anti-FVIII antibodies in hemophilic mice after treatment with human FVIII could be prevented completely by a blockade of CD40/CD40L interactions using MR1. Furthermore, FVIII-specific T-cell responses that included both Th1 and Th2 cells were suppressed when mice were treated with FVIII and MR1. The initial blockade of CD40/CD40L interactions was, however, not sufficient to induce a lasting immune tolerance against FVIII. The immune suppression was abolished and both neutralizing anti-FVIII antibodies and FVIII-specific T cells developed when treatment with FVIII was continued after the omission of MR1. In addition, there were no alterations in the Th1/Th2 polarization induced by the initial blockade of CD40/CD40L interactions.

This month in J Lab Clin Med: Issue highlights for July 2000

This month in J Lab Clin Med: Issue highlights for July 2000

Acquired hemophilia A in women postpartum: clinical manifestations, diagnosis, and treatment.

Acquired hemophilia A in women postpartum is diagnosed by a prolonged activated partial thromboplastin time (APTT), low plasma levels of coagulant factor VIII, and the detection of an inhibitor against factor VIII in the Bethesda assay. Effective treatment of bleeding symptoms should be based upon the clinical situation and depends on the inhibitor characteristics against human and porcine factor VIII. Immunosuppression usually does not significantly affect the disappearance of the factor VIII inhibitor antibody. The natural history of acquired hemophilia postpartum is independent of immunosuppressive treatment and featured by spontaneous disappearance of the inhibitor against factor VIII in the majority of cases.

Acquired inhibitory body hemophilia

Although autoantibodies against factor VIII or factor IX are a rare phenomenon, they are associated with a high risk of bleeding and high mortality. The condition, termed acquired haemophilia, occurs equally in both sexes and is most frequent in higher age groups. Patients typically present with severe bleedings in muscles and skin. In contrast to patients with congenital haemophilia and inhibitors, joint bleedings are very rare. In approximately half of all cases an associated disease state can be identified as the cause of autoantibody formation. An immediate and comprehensive diagnosis is essential for a rapid initiation of therapy. Equally important are a careful diagnostic differentiation between congenital and acquired factor deficiencies and the exclusion of non-specific inhibitors, which increase the occurrence of thrombolic events. The inhibitor titre, quantified using the Bethesda assay, is an important criterion for selecting the appropriate therapy. A wide range of treatment options is now available for the management of bleedings in patients with acquired haemophilia, namely porcine factor VIII, recombinant factor VIIa, prothrombin complex concentrates, and immunoadsorptions. In addition, immunosuppressive therapies are used to achieve permanent reduction or elimination of inhibitors.

Factor VIII inhibitors. Laboratory diagnosis of inhibitors.

The diagnosis of inhibitors of blood coagulation is often the most challenging problem in the clinical laboratory. Immediate attention must be given to the following patient groups whose principal laboratory abnormality is the prolonged activated partial thromboplastin time (aPTT): the patient with (1) hemophilia who previously responded to an adequate dose of clotting factor product and now fails to show effective clinical response to the same replacement concentrate; (2) previously benign clinical history who now presents with soft tissue bleeding or emergent internal hemorrhaging; (3) sudden onset of generalized ecchymoses who was previously well; (4) postpartum state; (5) malignancy, lymphoma, rheumatoid arthritis, or other autoimmune disorders; and (6) drug reactions. Immediate attention must be given to the prolonged prothrombin time (PT), aPTT, and thrombin time (TT) in order to respond to urgent queries from a perplexed internist, hematologist, intensivist, or surgeon caring for a patient with unexpected bleeding. Sometimes the problem of a prolonged "clotting time" arises preoperatively, causing unanticipated delay in operative procedures. For this reason, the laboratory support, usually in the coagulation section of a clinical laboratory or reference laboratory, must be quick, unequivocal and precise. The most common finding is an isolated mild, moderate, or severe prolongation of the aPTT with a normal PT, TT, and platelet count. The aPTT mixing study (The Mix), usually modified for time and temperature, along with appropriate controls, is the seminal test. This is the basis for all further testing. It may be supported by direct factor assays, and, therefore, the laboratory must know the reagent responsiveness and sensitivity for each clotting factor. By definition, complete correction of the aPTT in a 1:1 mix of patient and reference plasma is a factor deficiency. In this article, incomplete or minimal correction of The Mix will be characterized with particular attention to the various inhibitor assays, in other words, Oxford, Bethesda, and Nijmegen assays and the enzyme-linked immunosorbent assay (ELISA). An investigative approach to final characterization of the intensity (quantification) of the inhibitor and the exclusion of a lupus anticoagulant (LA) will be discussed.

Persistent expression of canine factor IX in hemophilia B canines.

We previously demonstrated that direct intramuscular injection of recombinant adeno-associated virus (rAAV) carrying the human FIX (hFIX) cDNA can safely be administered to hemophilic B canines and express human factor IX protein; however, the functional activity of the hFIX protein could not be assessed due to anti-human FIX antibody (inhibitor) formation. To test the therapeutic efficacy of rAAV in hemophilic dogs, rAAV type 2 (rAAV2) carrying canine FIX (cFIX) cDNA was injected into the skeletal muscle of two dogs at doses of 1012-13particles. Circulating cFIX protein levels were maintained for 1 year at levels of 1-2% of normal. Hemostatic correction (WBCT and APTT) paralleled plasma FIX antigen levels. Both dogs still required plasma infusion for spontaneous and traumatic bleeding events. Inhibitors to cFIX protein were not detected in either animal by Bethesda assay. Neutralizing antibodies directed against AAV-2 capsid were pronounced and persistent. Vector DNA and mRNA transcripts were detected only at the injected skeletal muscle tissue. Analysis of both high and low molecular weight DNA identified both replicative episomal and integrated AAV species. These results demonstrate that persistent secretion of the FIX transgene protein, necessary for successful gene therapy of hemophilia B, can be achieved using the parvovirus-based rAAV vector

Human Inhibitor Antibodies Specific for the Factor VIII A2 Domain Disrupt the Interaction between the Subunit and Factor IXa

Factor VIIIa, a heterotrimer of the A1, A2, and A3-C1-C2 subunits, increases the catalytic efficiency for factor IXa-catalyzed activation of factor X. A significant fraction of naturally occurring, anti-factor VIII inhibitor antibodies reacts with the A2 domain. Utilizing the capacity for isolated A2 subunit to stimulate factor IXa activity, we show that a panel of these inhibitors block this activity. Inhibition of activity parallels the antibody potency as measured in the Bethesda assay. These antibodies also block the A2-dependent increases in fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa. Similar to the IgG fractions, a peptide representing the sequence of the inhibitor epitope (A2 residues 484-509) blocked the A2-dependent stimulation of factor IXa. These results indicate that antibodies possessing this specificity directly inhibit the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this subunit to cofactor activity. Furthermore, these results also suggest that factor VIII residues 484-509 contribute to a factor IXa-interactive site.

DEVELOPMENT OF AUTOANTIBODIES AND FACTOR VIII INHIBITOR IN AN HIV‐INFECTED HAEMOPHILIAC FOLLOWING TREATMENT WITH COMBINATION ANTI‐RETROVIRAL THERAPY

The benefit of protease inhibitor therapy in HIV-infected haemophiliacs has been summarized recently ( 4). We have observed a variety of immunological complications following immune reconstitution in a patient treated successfully with protease inhibitors. A 14-year-old boy, diagnosed at birth in 1983 with severe haemophilia A (factor VIII level < 0.0 iu/dl), was treated on demand with factor VIII replacement until 1989 and prophylactic replacement thereafter (25 units/kg three times weekly). Between 1985 and 1991 he received intermediate purity factor VIII (NHS 8Y, BPL, U.K.) and, from 1991, high-purity factor VIII (8SM/Replenate, BPL, U.K.). He was found to be antibody positive for human immunodeficiency virus in 1985 and for hepatitis C in 1991. In 1988 he developed immune thrombocytopenia which was refractory to steroids but splenectomy produced a complete sustained remission. In 1991, when his CD4 count was 0.4 × 109/l, he developed HIV encephalopathy and commenced zidovudine with improvement in cognitive function. In 1997 he was admitted for investigation of pyrexia of unknown origin. No infective or neoplastic cause could be found. However, with a CD4 count below the limit of detection and a HIV viral load of 160 000/ml, he was converted to combination antiretroviral therapy with the addition of lamivudine and the protease inhibitor indinavir. Six months later his CD4 count had risen to 1.76 × 109/l and HIV RNA was undetectable in peripheral blood by nucleic acid sequence based amplification. At this time the frequency of spontaneous joint bleeds increased and a poor clinical response to factor VIII infusion was observed. A factor VIII inhibitor was quantified at 4 Bethesda units (BU). His prophylactic factor VIII replacement was increased to 50 units/kg three times weekly as low-dose immunotolerance and acute bleeding episodes were treated with FEIBA (Immuno, U.K.). 6 weeks later he developed bleeding from venepuncture sites and spontaneous muscle bleeds. The factor VIII inhibitor had risen to 57 BU. A full blood count revealed Hb 7.6 g/dl, WBC 8.3 × 109/l. neutrophils 0.2 × 109/l and platelet count 6 × 109/l. The direct antiglobulin test (DAGT) was positive. A bone marrow examination was not performed, but 3 d after administration of intravenous immunoglobulin 1 g/kg/d for 2 d and prednisolone 1 mg/kg/d his platelet count rose to 197 × 109/l, indicating a probable diagnosis of immune thrombocytopenia (ITP). Concurrently his neutrophil count rose to 1.49 × 109/l after 5 d and his DAGT became negative. In view of his severe haemorrhagic tendency and rapidly rising inhibitor titre the patient was commenced on a high-dose immune tolerance programme with 100 units/kg twice daily factor VIII (NHS 8Y) ( 2). After only 10 d of this regimen the inhibitor became undetectable in the Bethesda assay. Over the subsequent weeks his factor VIII recovery normalized, and 8 weeks after starting the tolerance programme a factor VIII half-life study was normal (t1/2 = 17 h). His factor VIII replacement has now been reduced to 25 units/kg three times weekly. Factor VIII inhibitors occur in approximately a quarter to a third of patients with severe congenital factor VIII deficiency after a median of 10 treatment days ( 3). Our patient is unusual as he developed an inhibitor to factor VIII after > 300 treatments and without a recent change in factor concentrate. He also developed other immune phenomena: ITP, autoimmune haemolysis, and possibly autoimmune neutropenia. All responded extremely rapidly to therapy and have not subsequently relapsed. These observations raise the possibility that immune reconstitution due to successful combination anti-retroviral therapy resulted in de novo autoimmune phenomena and an inhibitor to factor VIII. As 20% of HIV-positive individuals develop immune thrombocytopenia it is impossible to know if our patient's previous thrombocytopenia was indicative of an underlying predisposition to autoimmunity. However, in patients with haemophilia who receive factor concentrate the likelihood of alloantibody formation following immune reconstitution may be similar to that following initial exposure to factor VIII. There has been concern that the use of protease inhibitors leads to an increased bleeding tendency in haemophiliacs. However, this has not usually been associated with the development of a factor VIII inhibitor. To our knowledge there has been only one case of a new inhibitor developing in a heamophiliac receiving the same combination of anti-retrovirals as the patient described here ( 1). Patients with haemophilia treated in this way may be susceptible to inhibitor development, but our observation suggests this may be transient or easily eliminated with a desensitization regime.

Surveillance for Factor VIII Inhibitor Development in the Canadian Hemophilia A Population Following the Widespread Introduction of Recombinant Factor VIII Replacement Therapy

In the Fall of 1994 the majority of Canadian Hemophilia A (Factor VIII (F.VII) deficiency) patients who were receiving replacement therapy were converted to Recombinant Factor VIII (rF.VIII) from plasma derived products. This decision was taken and funded by the Canadian Blood Agency following the advice of the Association of Hemophilia Centre Directors of Canada (AHCDC) who considered this to be the safest replacement therapy available. Although it was the considered opinion of the AHCDC that there was no evidence available to support the theoretical concern that rF.VIII may prove more immunogenic than plasma derived products, patient follow up included intensive surveillance of all patients converted for this complication. A central reference laboratory was established and plasma specimens obtained before and 6–12 months following conversion to rF.VIII were referred for evaluation for inhibitor development by the classical Bethesda Assay. By concensus of the referring centers a Bethesda Unit (BU)/activity of 0.5 or greater was considered to be clinically significant. No increase in the incidence of inhibitor development has been recorded in 478 patients followed for one year after conversion. This pattern has not changed in 339 of these patients followed for a further year. Of interest was the finding by the reference laboratory that 8.0% of patients had BU activity of 0.5 or greater before conversion to rF.VIII. Many of these individuals lost this activity after conversion to rF.VIII as did others who appeared to have developed inhibitory activity during the first year of follow up but became inhibitor free in the second year of therapy. Overall, the incidence of true inhibitor development, i.e. negative pre-/positive post-conversion to rF.VIII, in this population of Hemophilia A patients was 2–3% over 2 years. This is similar to the incidence in patients treated with plasma derived products. These data emphasize the need for rigorous baseline evaluation in such investigations and the heterogeneity of the inhibitor response as assessed by in vitro assay. It was concluded that, although no attempt was made to correlate these in vitro data with clinical observations including F.VIII recovery and survival, the use of rF.VIII in hemophiliacs previously treated with plasma derived products was not associated with an increase in F.VIII inhibitor development.

A Detailed Comparison of the Performance of the Standard versus the Nijmegen Modification of the Bethesda Assay in Detecting Factor VIII:C Inhibitors in the Haemophilia A Population of Canada

The Bethesda assay is widely used to monitor the development and progress of Factor VIII:C inhibitors. Factor VIII stability in the substrate plasma (normal pool) is compromised by pH shift and reduction in protein concentration. Preliminary study, by Verbruggen and colleagues (8), suggested a reduction in spuriously positive assay results may result from buffering the normal pool plasma substrate with imidazole to pH 7.4 and substituting Factor VIII deficient plasma for imidazole buffer in the control incubation mix. These laboratory findings have now been confirmed by the performance of both the standard and the modified Bethesda assays in parallel on 877 patient samples screened during the Factor VIII:C Inhibitor Surveillance Program instituted following the conversion of all Canadian haemophilia A patients to recombinant Factor VIII. Although this study does not address the question of the clinical significance of spurious positive assays, these laboratory findings do support the conclusions of Verbruggen and the modified assay has recently been endorsed by the Factor VIII/IX Subcommittee of the SSC.

Analysis of the Human Factor VIII A2 Inhibitor Epitope by Alanine Scanning Mutagenesis

Antibodies directed to the A2 domain of factor VIII (fVIII) are usually an important component of the polyclonal response in patients who have clinically significant inhibitory antibodies to fVIII. A major determinant of the A2 epitope has been located by homolog scanning mutagenesis using recombinant hybrid human/porcine fVIII molecules to a sequence bounded by Arg484-Ile508 (Healey, J. F. , Lubin, I. M., Nakai, H., Saenko, E. L., Hoyer, L. W., Scandella, D. , and Lollar, P. (1995) J. Biol. Chem. 270, 14505-14509). Within this region, human residues Arg484, Pro485, Tyr487, Ser488, Arg489, Pro492, Val495, Phe501, and Ile508 differ from porcine fVIII. We stably expressed in mammalian cells nine active B-domainless human fVIII molecules containing single alanine substitutions at these sites. Their inhibition by a murine anti-A2 monoclonal antibody, monoclonal antibody (mAb) 413, and by three A2-specific alloimmune and two A2-specific autoimmune human inhibitor plasmas was measured by the Bethesda assay. The inhibition of Arg484 --> Ala, Tyr487 --> Ala, Arg489 --> Ala, and Arg492 --> Ala by mAb413 was reduced by greater than 90% compared with wild-type, B-domainless human fVIII. mAb413 inhibited the most severely affected mutant, Arg489 --> Ala, 0.01% as well as wild-type fVIII. For all five patient plasmas, the Tyr487 --> Ala mutant displayed the greatest reduction in inhibition. The inhibition of the Tyr487 --> Ala mutant by these antibodies ranged from 10% to 20% that of wild-type fVIII. The inhibition of the Ser488 --> Ala, Arg489 --> Ala, Pro492 --> Ala, Val495 --> Ala, Phe501 --> Ala, and Ile508 --> Ala mutants by most of the plasmas also was significantly reduced. In contrast, the Arg484 --> Ala and Pro485 --> Ala mutants were relatively unaffected. Thus, although mAb413 binds to the same region as human A2 inhibitors, it recognizes a different set of amino acid side chains. The side chains recognized by human A2 inhibitors appear to be similar, despite the differing immune settings that give rise to fVIII alloantibodies and autoantibodies.

Adsorption of immunoglobulin G and anti-Factor VIII inhibitory antibody from haemophiliac plasma to derivatized polystyrenes

Modified polystyrene resins containing sulphonate groups and tyrosyl sulphamide or tyrosyl methyl ester sulphamide groups have been investigated with respect to their potential for selective binding of anti-Factor VIII inhibitory antibodies from plasma. Adsorption of total immunoglobulin G and of a monoclonal antibody to Factor VIII was measured following addition of the radioiodinated proteins to normal plasma, plasma depleted of Factor VIII by adsorption on a resin coupled to anti-Factor VIM antibody, and haemophiliac plasma containing various levels of inhibitory anti-Factor VIII antibody. Depletion of anti-Factor VIII antibody from the haemophiliac plasmas by incubation with the resins was also measured by Bethesda assay. The modified resins and their corresponding unmodified ‘controls’ showed similar binding of total immunoglobulin G. However, only resins containing either sulphonate or a combination of sulphonate and tyrosyl sulphamide groups showed evidence of selective adsorption of anti-Factor VIII antibody from plasma.

High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the half-life of transfused FVIII.

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post-transfusion antibodies may also arise against non-coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII-induced antibodies by use of an enzyme-linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non-inhibitory antibodies was evaluated in three patients; although half-life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti-FVIII antibodies by ELISA when routinely assessing haemophilia A patients.

9 Acquired inhibitors

Factor VIII auto-antibody inhibitors, though rare, may present significant and often life-threatening haemorrhage. These auto-antibodies, arising predominantly in older individuals, occur in association with autoimmune disorders, lymphoproliferative disorders, solid tumours, medications and the postpartum state. Almost half of the patients develop auto-antibodies spontaneously without an underlying medical condition. Factor VIII auto-antibody inhibitors are characterized as polyclonal IgG immunoglobulins directed against the FVIII procoagulant activity. Laboratory diagnosis is made by performing the aPTT clotting time in conjunction with a mixing study, and subsequently with specific factor assays. Auto-antibodies are quantified most commonly utilizing the Bethesda assay. Acquired inhibitors to other coagulation factors, including factors IX, XI, XIII, vWF protein, and the vitamin K-dependent proteins are extremely rare. The principles of therapy are similar to those which apply to the management of factor VIII auto-antibodies. Treatment of patients with acquired factor VIII auto-antibody inhibitors varies depending upon the underlying medical condition, the titre of the inhibitor, and the clinical presentation. Acutely bleeding patients with high-titre auto-antibodies generally respond well with infusions of porcine factor VIII concentrate, PCCs or rFVIIa. Extracorporeal plasmapheresis with exchange will acutely reduce circulating antibodies and can be used in conjunction with factor infusions and/or IgIV. Haemorrhage in a patient with a low titre auto-antibody will usually respond to high doses of human factor VIII concentrate. DDAVP may also increase factor VIII levels in patients with low-titre inhibitors. Long-term reduction of auto-antibodies can be achieved by immuno-suppressive regimens using steroids and/or cytotoxic agents, IgIV and interferon-alpha. The selection of the appropriate treatment depends upon the associated medical condition, likelihood of spontaneous remission, risk of toxicities of therapy and cost. Determining the efficacy and safety of new treatment modalities for factor VIII auto-antibodies and other coagulation factor inhibitors will require multicentre randomized clinical trials.

Recommendations for the treatment of factor VIII inhibitors

A strategy is described for the initial detection, management and elimination of factor VIII inhibitors arising in patients with congenital and acquired haemophilia A. It is suggested that children with severe haemophilia A should be screened every 3 months up to the age of 10 years for inhibitors using the Bethesda method. Factor VIII inhibitors arising in these patients should be abolished using immune-tolerance induction wherever possible. Such regimes should be started as early as possible, preferably when the inhibitor titre is < 10 Bethesda Units (BU)/ml, and should not be interrupted. High-intensity regimes are recommended for patients whose inhibitors exceed 10 BU. Autoantibodies to factor VIII giving rise to acquired haemophilia should be abolished using high-dose immunoglobulin or conventional immunosuppression. The choice of haemostatic agent for the treatment of severe bleeding should be based upon the clinical circumstances and the current inhibitor value, measured using both human and porcine factor VIII in the Bethesda assay. The past anamnestic response should also be considered when choosing treatment for minor bleeding episodes.

The effect of head elevation on cerebral perfusion pressure in fulminant hepatic failure: [formula omitted], Depts. of Medicine, Neurosurgery and Neurology, Jefferson Medical College, Philadelphia, PA

The inhibitor titer is the most important clinical measurement in inhibitor patients, and the Nijmegen method is preferable to the original and well-established Bethesda assay for this purpose; however, both methods have high inter-laboratory variability. Monitoring inhibitor patients after treatment with bypassing agents is difficult. Treatment with recombinant activated factor VII (rFVIIa; NovoSeven®, Novo Nordisk, Bagsværd, Denmark) can be monitored with assays for FVII clotting activity or with a specific assay for FVIIa; while the latter is more reproducible, its relevance to the clinical response of individual patients remains unclear. Recent years have also witnessed a revival of global assays, which contribute useful additional information and which may be more relevant to the hemostatic state of individual inhibitor patients. One such assay currently undergoing a renaissance is the thrombin generation test (TGT). The TGT has many methodological variations—several of which have been studied in our laboratory—and shows the most promise for use in treatment monitoring. This article reviews the assays most appropriate for monitoring inhibitor patients and discusses some of the most recent developments in the use of global assays in this indication.

The Nijmegen Modification of the Bethesda Assay for Factor VIII:C Inhibitors: Improved Specificity and Reliability

Antibodies against factor VIII coagulant activity can appear in haemophiliacs who are treated with factor VIII preparations but also spontaneously in non-haemophiliacs. The Bethesda assay is the most commonly used method to detect these antibodies, but it lacks specificity especially in the lower range resulting in unreliable data. Two modifications are proposed and tested to resolve the imperfections: 1. Buffering the normal plasma used in the assay- and control mixture with 0.1 M imidazole to pH 7.4. 2. Replacing the imidazole buffer in the control mixture by immunodepleted factor VIII deficient plasma. These modifications allow better discrimination between positive and negative samples and improve reliability.