Summary
Background
The development of neutralizing antibodies, referred to as inhibitors, against factor VIII is a major complication associated with FVIII infusion therapy for the treatment of ...hemophilia A (HA). Previous studies have shown that a subset of HA patients and a low percentage of healthy individuals harbor non‐neutralizing anti‐FVIII antibodies that do not elicit the clinical manifestations associated with inhibitor development.
Objective
To assess HA patients' anti‐FVIII antibody profiles as potential predictors of clinical outcomes.
Methods
A fluorescence immunoassay (FLI) was used to detect anti‐FVIII antibodies in 491 samples from 371 HA patients.
Results
Assessments of antibody profiles showed that the presence of anti‐FVIII IgG1, IgG2 or IgG4 correlated qualitatively and quantitatively with the presence of an FVIII inhibitor as determined with the Nijmegen–Bethesda assay (NBA). Forty‐eight patients with a negative inhibitor history contributed serial samples to the study, including seven patients who had negative NBA titers initially and later converted to being NBA‐positive. The FLI detected anti‐FVIII IgG1 in five of those seven patients prior to their conversion to NBA‐positive. Five of 15 serial‐sample patients who had a negative inhibitor history and had anti‐FVIII IgG1 later developed an inhibitor, as compared with two of 33 patients with a negative inhibitor history without anti‐FVIII IgG1.
Conclusions
These data provide a rationale for future studies designed both to monitor the dynamics of anti‐FVIII antibody profiles in HA patients as a potential predictor of future inhibitor development and to assess the value of the anti‐FVIII FLI as a supplement to traditional inhibitor testing.
Essentials
Immunologic methods detect factor VIII (FVIII) antibodies in some inhibitor‐negative specimens.
Specimens were tested by modified Nijmegen‐Bethesda assay (NBA) and fluorescence ...immunoassay.
The NBA with preanalytical heat inactivation detects FVIII inhibitors down to 0.2 NBU.
IgG4 frequency validates the established threshold for positivity of ≥ 0.5 NBU for this NBA.
Summary
Background
The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25–75% residual activity (RA), corresponding to 0.4–2.0 Bethesda units, with the use of ‘more sensitive methods’ for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen‐Bethesda assay NBA) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with sensitive immunologic methods.
Objective
To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)‐modified NBA (CDC‐NBA), which includes preanalytic heat inactivation to liberate bound anti‐FVIII antibodies.
Methods
Specimens with known inhibitors were tested with the CDC‐NBA. IgG4 anti‐FVIII antibodies were measured by fluorescence immunoassay (FLI).
Results
Diluted inhibitors showed linearity below 0.4 Nijmegen‐Bethesda units (NBU). With four statistical methods, the limit of detection of the CDC‐NBA was determined to be 0.2 NBU. IgG4 anti‐FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥ 0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU.
Conclusions
The CDC‐NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti‐FVIII IgG4 in some patients with negative (< 0.5) NBU. The sharp increase in IgG4 frequency between 0.4 and 0.5 NBU validates the established threshold for positivity of ≥ 0.5 NBU for the CDC‐NBA, supporting the need for method‐specific thresholds.
The relationships between primary human immunodeficiency virus type 1 (HIV-1) Gag-specific cytotoxic T lymphocyte (CTL) frequency, virus load, and CD4 T cell loss were evaluated in a group of 46 ...HIV-1-infected persons with hemophilia. Freshly isolated peripheral blood mononuclear cells in limiting dilution assays were used to measure HIV-1 Gag-specific CTL frequencies. Concurrent measurements of virus load and lymphocyte surface markers were obtained. No correlation between Gag-specific CTL frequency and concurrent CD4 cell count was observed. A significant inverse relationship was observed between HIV-1 Gag-specific CTL frequency and provirus load as measured by polymerase chain reaction. Subjects with higher CTL frequencies were found to have more stable CD4 cell counts over time. These results provide additional evidence to support the concept that the predominant role of this virus-specific cellular immune response is to limit viral replication and CD4 cell loss in HIV-1 infection.
Seven long-term nonprogressors (LTNPs) have been identified in a cohort of 128 human immunodeficiency virus (HIV)-1 infected individuals with hemophilia. Studies included quantitation of virus by ...polymerase chain reaction, characterization of primary virus isolates in vitro, analysis of lymphocyte surface markers, and measurement of virus-specific cytotoxic T lymphocytes (CTLs). Viruses of LTNPs exhibited slow growth in vivo and in vitro. LTNPs had expansion of CD8 T cells with increased expression of HLA-DR. Intermittent HIV-1—specific CTL effector activity was detected in freshly isolated peripheral blood mononuclear cells of most LTNPs. CTL precursor frequencies were higher in LTNPs than in patients with progressive disease. Virus antigen—specific lymphoproliferation was vigorous in some LTNPs. Thus, LTNPs in this cohort have maintained remarkably low virus burdens and vigorous HIV-1—specific cell-mediated immunity over a 15-year period. The presence of expanded, activated CD8 T cells with cytotoxic effector function in the peripheral blood suggests ongoing viral replication.
Essentials
Studies characterizing neutralizing antibodies (inhibitors) in hemophilia B (HB) are lacking.
The current study describes anti–factor (F) IX antibody profiles in 37 patients who have HB.
...Anti‐FIX IgG4 levels exhibited a strong positive correlation with Nijmegen–Bethesda results.
These data will help to more clearly define, predict, and treat alloantibody formation in HB.
Summary
Background
Hemophilia B (HB) is an inherited bleeding disorder caused by the absence or dysfunction of coagulation factor IX (FIX). A subset of patients who have HB develop neutralizing alloantibodies (inhibitors) against FIX after infusion therapy. HB prevalence and the proportion of patients who develop inhibitors are much lower than those for hemophilia A (HA), which makes studies of inhibitors in patients with HB challenging due to the limited availability of samples. As a result, there is a knowledge gap regarding HB inhibitors.
Objective
Evaluate the largest group of patients with inhibitor‐positive HB studied to date to assess the relationship between anti‐FIX antibody profiles and inhibitor formation.
Methods
A fluorescence immunoassay was used to detect anti‐FIX antibodies in plasma samples from 37 patients with HB.
Results
Assessments of antibody profiles showed that anti‐FIX IgG1‐4, IgA, and IgE were detected significantly more often in patients with a positive Nijmegen–Bethesda assay (NBA). All NBA‐positive samples were positive for IgG4. Anti‐FIX IgG4 demonstrated a strong correlation with the NBA, while correlations were significant, yet more moderate, for anti‐FIX IgG1‐2 and IgA.
Conclusions
The anti‐FIX antibody profile in HB patients who develop inhibitors is diverse and correlates well with the NBA across immunoglobulin (sub)class, and anti‐FIX IgG4 is particularly relevant to functional inhibition. The anti‐FIX fluorescence immunoassay may serve as a useful tool to confirm the presence of antibodies in patients who have low positive NBA results and to more clearly define, predict, and treat alloantibody formation against FIX.
Summary
Background
Hemophilia A (HA) is an X‐linked bleeding disorder caused by a deficiency in factor VIII (FVIII). von Willebrand disease (VWD) is characterized by a quantitative or qualitative ...defect in von Willebrand factor (VWF). Patients with VWD with severely low VWF or VWD Type 2N (VWD2N), a VWD subtype distinguished by defective VWF binding to FVIII, may have reduced FVIII levels secondary to their VWD. These patients superficially resemble patients with HA and pose a potential for misdiagnosis.
Objectives
To investigate the unexplained cause of bleeding in HA patients without known FVIII mutations by assessing plasma VWF antigen (VWF:Ag), FVIII binding capacities and VWF genotypes.
Patients/Methods
Thirty‐seven of 1027 patients with HA studied as part of the Hemophilia Inhibitor Research Study lacked identifiable F8 mutations. These patients (cases) and 73 patients with identified F8 mutations (controls) were evaluated for VWF:Ag, a patient's VWF capacity to bind FVIII (VWF:FVIIIB) and VWF sequence.
Results
Four cases had VWF:Ag < 3 IU dL−1 and VWF mutations consistent with Type 3 VWD. Six cases and one control were heterozygous for mutations previously reported to cause Type 1 VWD (VWD1) (n = five cases and one control) or predicted to be deleterious by Polyphen2 and SIFT prediction tools (n = 1 case). One control had VWF:Ag < 30 IU dL−1 and seven patients (four cases and three controls), including two cases who were heterozygous for a known VWD2N mutation, had reduced VWF:FVIIIB.
Conclusions
These data emphasize that some patients diagnosed with HA require VWF assessments in order to achieve a comprehensive diagnosis and an optimal treatment strategy.
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