1). The median age was 14.0 years (mean: 17.3 years, range: 0–74 years), and the majority of patients were Caucasians (84.1%). In addition, there were nine Hispanics, two Asians,

eight of African descent and 13 of other ethnic origins. The disease-causative mutation was identified in 190 patients (94.5%), with 110 patients carrying an inversion mutation, seven patients a large deletion, 17 patients a nonsense mutation, 36 patients a small deletion/insertion, two patients a splice-site mutation and 18 patients a missense mutation. Seventy-nine (39.3%) patients were reported to have a history of an inhibitor (see Fig. 1), but had no current titre, with a median historical peak titre of 13.0 BU mL−1 (mean: check details 152.4 BU mL−1, range: 1.0–3000 BU mL−1). The majority (68.4%) of the subjects were high-responders, ranging click here from 5.0 to 3000 BU mL−1 (median: 34.0 BU mL−1, mean: 216.3 BU mL−1). ITI was initiated in 83.5% (66/79) of these patients, and the treatment was reported to be successful in 89.4% (59/66) of them. Three patients were undergoing ITI at the time of plasma sample collection, and failure of ITI was reported in four patients. Eight (8/78; 10.3%) families were concordant for a history of positive inhibitor titres in all siblings, 53 families (67.9%) were discordant and 17 families (21.8%)

were concordant for no history of inhibitory FVIII antibodies. The immunoassay was performed using three different commercially available recombinant FVIII concentrates:

the full-length recombinant selleck products preparations Advate® (Baxter, Deerfield, IL, USA) and Kogenate® (Bayer AG, Leverkusen, Germany), and the recombinant B-domain-deleted ReFacto® (Wyeth, Maidenhead, UK). The FVIII concentrates were used in separate solutions with a final FVIII concentration of 2 μg mL−1, and also in a mixture where all three concentrates were combined to reach the same final concentration, 2 μg mL−1, of FVIII-antigen. Microtitre plates (Nunc-Immunoplate, Roskilde, Denmark) were plated with 50 μL per well of FVIII-solution (2 μg mL−1) and incubated at 4°C overnight. The plates were washed three times each with washing buffer (0.05 m Tris–HC1, 0.15 m NaCl, pH 7.5 with 0.1% Tween 20) using a plate washer (Nunc-Immuno Wash before non-specific sites were blocked by adding 100 μL of 1% bovine serum albumin (BSA; ICN Biomedicals inc., Irvine, CA, USA) in quench buffer (0.05 m Tris–HC1, 0.15 m NaCl, pH 7.5 with 1% BSA). The plates were incubated for 60 min at room temperature. After incubation, the plates were washed three times each with washing buffer. Patient plasma samples were thawed at 37°C for 5 min and diluted in quench buffer to 1/100 before 50 μL of the sample was plated in duplicate on the microtitre plate.