Efficacy, safety and tolerability of recombinant factor VIII (REFACTO) in patients with haemophilia A: interim data from a postmarketing surveillance study in Germany and Austria.

An open-label, multicentre, postmarketing surveillance study conducted in Germany and Austria with recombinant factor VIII (REFACTO) has enrolled 217 patients (mean age 26.3 years) from 38 haemophilia centres during the first 4.8 years. Most patients (188/217; 86.6%) had severe to moderately severe haemophilia A, of whom 153 completed sufficient diary information for the main efficacy analysis. These 153 patients experienced a median of 6.6 (interquartile range 1.4-18.6) bleeding episodes per year. Patients treated with prophylaxis experienced a median of 4.4 (1.1-9.3) bleeds per year, while patients treated on-demand experienced a median of 22.8 (11.3-29.0) bleeds per year. Overall, most physicians (41/43 [95.3%]) were 'very satisfied' or 'satisfied' with the efficacy of REFACTO in the treatment of bleeding episodes. A total of 137 non-serious adverse events have been reported in 52/217 patients (24.0%) to date. In addition, 129 serious adverse events in 87 patients (40%) were reported, including 41 cases of 'less than expected therapeutic effect' (LETE). Of these, 39 LETE cases were reported in one centre; however, patients in this centre experienced considerably fewer bleeding episodes per year than patients outside this centre. Overall, six patients (2.8%) have developed de novo inhibitors, three of which were considered high titre. Four of these patients were at high risk (0-50 exposure days [ED]) of inhibitor formation, one was at intermediate risk (51-100 ED) and one was at low risk (>100 ED). These results emphasize the benefit of postmarketing surveillance and, overall, this study confirms the efficacy, safety and tolerability of REFACTO in the treatment of patients with haemophilia A.

Correlations between antibody immune responses at different mucosal effector sites are controlled by antigen type and dosage.

Monitoring specific secretory immunoglobulin A (IgA) responses in the intestines after mucosal immunization or infection is impeded by the fact that sampling of small intestinal secretions requires invasive methods not feasible for routine diagnostics. Since IgA plasma cells generated after intragastric immunization are known to populate remote mucosal sites as well, secretory IgA responses at other mucosal surfaces may correlate to those in the intestines and could serve as proxy measures for IgA secretion in the gut. To evaluate the practicability of this approach, mice were immunized intragastrically with 0.2, 2, and 20 mg of ovalbumin plus 10 microg of cholera toxin, and the antigen-specific local secretory IgA responses in duodenal, ileal, jejunal, rectal, and vaginal secretions, saliva, urine, and feces, as well as serum IgG and IgA responses were analyzed by enzyme-linked immunosorbent assay. Correlation analysis revealed significant relationships between serum IgG and IgA, urinary IgA, salivary IgA, and secretory IgA in duodenal, jejunal, ileal, and rectal secretions for the 0.2-mg but not for the 20-mg ovalbumin dose. Fecal samples were poor predictors for intestinal antiovalbumin IgA responses, and no correlations could be established for cholera toxin, neither between local anti-cholera toxin levels nor to the antiovalbumin responses. Thus, specific IgA in serum, saliva, or urine can serve as a predictor of the release of specific IgA at intestinal surfaces after intragastric immunization, but the lack of correlations for high ovalbumin doses and for cholera toxin indicates a strong dependency on antigen type and dosage for these relationships.

A stable and highly sensitive 3,3',5,5'-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays.

3,3',5,5'-Tetramethylbenzidine (TMB) is a widely used chromogen for horseradish peroxidase-based detection systems because it yields reaction products with high absorption coefficients and lacks carcinogenicity. Unfortunately, TMB is labile and poorly soluble in aqueous buffers and such solutions must be freshly prepared before each experiment. Moreover, substrate depletion can occur under assay conditions. To overcome these problems we have developed a two-component TMB substrate system which has a lower detection limit and is more sensitive than many commercially available TMB reagents when compared in microtiter plate enzyme-linked immunosorbent assays. Both components of the substrate system are stable for at least 1 year at 4 degrees C and the usual discoloration of TMB stock solutions is prevented by the addition of a stabilizer that decomposes upon mixing of the two components.