A longitudinal evaluation of anti-FVIII antibodies demonstrated IgG4 subclass is mainly correlated with high-titre inhibitor in haemophilia A patients.

The development of inhibitory antibodies against factor VIII (FVIII) (inhibitor) is the major complication in haemophilia A patients. The FVIII-binding antibodies development comprises a polyclonal immunoglobulin (Ig) G response. Recent studies showed strong correlation between the presence of neutralizing anti-FVIII antibodies (inhibitors) and IgG4 subclass. The aim of this study was to evaluate anti-FVIII IgG subclasses in haemophilia A patients with inhibitor both in a cross-sectional and in a longitudinal analysis. Inhibitors were determined by Nijmegen-Bethesda assay. Anti-FVIII IgG subclasses were performed by ELISA, and samples from 20 healthy individuals were used to validate the test. We studied 25 haemophilia A patients with inhibitor, previously treated exclusively with plasma-derived FVIII concentrates or bypassing agents. The IgG subclasses distributions were evaluated in two groups of patients classified according to inhibitor response. IgG1 and IgG4 antibodies were most prominent in haemophilia A patients with inhibitors when compared with IgG2 and IgG3. This study reports for the first time the behaviour of FVIII-binding IgG1 and IgG4 subclasses in a longitudinal analysis, in a clinical setting, of high-response inhibitor haemophilia A patients, showing the correlation of IgG4 and the inhibitor titres. In spite of being considered a non-pathologic antibody subclass with anti-inflammatory properties in other situations, IgG4 is correlated with the presence of high-titre inhibitor in the haemophilia setting. The comprehension of the IgG4 role in immune response may be crucial to establish the process for designing specific tolerance to FVIII.

Allergic reaction in a cohort of haemophilia A patients using plasma-derived factor VIII (FVIII) concentrate is rare and not necessarily triggered by FVIII.

In contrast to haemophilia B, allergic manifestations are rare complications in haemophilia A (HA) patients treated with factor VIII (FVIII) concentrates. Nevertheless, it can be serious and hamper replacement therapy in these cases. The aims of this study were to evaluate the frequency of allergic reaction in a cohort of HA patients treated only with plasma-derived FVIII (pdFVIII) concentrates, and assess the possible immune mechanisms involved. History of allergic reaction was retrospectively assessed. Patients with allergic manifestations were followed, and had plasma samples collected in different timepoints in relation to the allergic episode. These samples were analysed for the presence of inhibitor and anti-FVIII immunoglobulins subclasses. Three of 322 HA patients (0.9%) developed allergic reaction after exposure to pdFVIII products during the last 15 years in our centre. The first patient, with severe HA, without inhibitor, had anti-pdFVIII IgE and IgG4, but no anti-recombinant FVIII (rFVIII) IgE. The second patient, with severe HA, and high-responding inhibitor, presented allergic manifestation with both, pdFVIII concentrate and activated prothrombin complex concentrate. Although anti-pdFVIII and anti-rFVIII IgG4 were detected, no anti-FVIII IgE was present. The third patient, with moderate HA without inhibitor, atopic, had no anti-FVIII immunoglobulin detected, and allergic symptoms disappeared after switching to rFVIII concentrate. This study corroborates the low incidence of allergic reactions in HA patients. In the three cases presented, the anti-FVIII immunoglobulin profile demonstrated that the allergic manifestation was triggered by other proteins contained in pdFVIII products, and not directed to FVIII.

Evaluation of the host response to endotoxemia of FVIII and FIX deficient mice.

For several years, coagulation has been implicated in the pathogenesis of sepsis. However, results from clinical trials with natural anticoagulants, as well as studies with knock-out mice for specific coagulation factors yielded conflicting results on the role of coagulation in the pathogenesis of sepsis. The aim of this study was to evaluate the impact of severe The factor VIII:C (FVIII:C) and factor IX:C (FIX:C) deficiency on a lipopolysaccharide (LPS)-induced murine model of sepsis. FVIII:C and FIX:C deficient mice, and their haemostatic normal littermate controls were challenged with LPS, and several parameters of the host response were evaluated: seven-day survival experiments were performed using two dose levels of LPS; biochemical and histological markers of tissue damage, coagulation parameters, and pro-inflammatory cytokines were evaluated at baseline and after 3 h and 6 h after an injection of LPS. Severe FVIII and FIX deficiency were compatible with normal survival in experimental sepsis. In addition, LPS-induced tissue damage and coagulation activation were similar in FVIII or FIX deficient mice compared to their respective controls. A lower release of pro-inflammatory cytokines was observed in FIX but not in FVIII deficient mice. Severe FIX or FVIII deficiency does not protect mice from mortality or from tissue damage in the endotoxemia model, supporting the hypothesis that FVIII and FIX are not critical to the pathogenesis of experimental sepsis.

Performance of a point-of-care device in determining prothrombin time in supra-therapeutic INRs.

INTRODUCTION: Point-of-care (POC) devices have been widely adopted for monitoring prothrombin time (PT) (INR) following the demonstration of their accuracy compared to standard INR determination. However, guidelines suggest confirmation of POC results when INRs increase above therapeutic range, due to concerns regarding possible inferior performance of POC devices in high INR levels. Unfortunately, patients with supra-therapeutic INRs are underrepresented in studies that validated these devices. METHODS: We performed a prospective evaluation of the performance of a POC device in monitoring oral anticoagulation in patients with INR values above 3.5 in a University outpatient anticoagulation clinic. During a 6-month period, 2322 INR determinations were performed with a POC device, and results above 3.5 were immediately repeated on an automated coagulometer. RESULTS: Dual INR determinations by two methods were obtained in 160 visits, with a mean INR from the POC device of 4.52 ± 0.96. Both classical statistics and clinical concordance analysis yielded satisfactory results when the two methods were compared. CONCLUSION: Our results demonstrate that POC devices present good correlation with standard laboratory methods for PT determination in supra-therapeutic INRs and that differences in clinical management do not support the need for systematic confirmation of these results in nonbleeding patients.