A higher result for plasma factor VIII:C measured by the one-stage as compared with the two-stage method has been described in some patients with haemophilia A or with von Willebrand's disorder. We used both methods to measure FVIII:C in 95 patients with haemophilia A. The results were equivalent in all 21 patients with severe haemophilia (16 families) and in 45 of the patients with mild or moderate haemophilia (18 families). However, the results were discrepant (FVIII:C by one-stage assay 2-7-fold higher than by two-stage assay) in the other 29 patients with mild or moderate haemophilia (12 other families). For each patient with discrepant FVIII:C results the classification was the same for all other affected members of his family. In some families with haemophilia A the gene defect leads to a discrepancy between the one-stage and two-stage FVIII:C results and may be more widespread than previously recognized.

A subgroup of patients with haemophilia A who have a familial discrepancy between the one-stage and two-stage factor VIII:C results has previously been described. These patients show factor VIII:C levels by one-stage assay that are 2-7-fold higher than their two-stage results. We have studied 10 such families and identified six different mutations in the factor VIII gene in this group. The chemical cleavage method and DNA sequencing was used to identify mutations in factor VIII gene fragments generated by reverse transcription and PCR. All available family members were tested to confirm the presence of the mutation in affected individuals. These patients were found to have one of six single point substitutions causing a missense mutation and alteration to one codon in exons 7, 11, 14 or 18. The mutations comprise three that have not previously been described (Ala284Glu. Arg698Leu. Leu1932Phe) and three that have been previously described (Ser289Leu, Arg531His, Arg698Trp). Alterations to the amino acid composition of the A1, A2 and A3 domains of factor VIII are predicted by these molecular studies. In contrast, a control group of 23 mild haemophilia families with equivalent factor VIII:C results by one-stage and two-stage assays did not have any of the above mutations. Detailed studies in seven of these latter families identified four mutations affecting the A3, C1 and C2 domains of factor VIII. These findings suggest a genetic basis to the unusual factor VIII phenotype but do not explain the mechanism of the discrepant factor VIII activity.

Summary. In some families with mild haemophilia higher results are obtained for factor VIII activity (FVIII:C) determined by one-stage assay than by two-stage or chromogenic assays. Amino-acid substitutions in the A1, A2 and A3 domains of factor VIII have been described in affected individuals with this phenotype. We describe a case of mild haemophilia A in which FVIII:C measured by one-stage assay was normal at 106%. However, FVIII:C levels measured by two-stage and chromogenic assays were 18% and 35% respectively. DNA analysis revealed a novel mutation in the A3 domain of factor VIII, His 1954 → Leu. In a molecular model of the FVIII A domains, His 1954 is placed in close proximity to two other mutations that have previously been shown also to be associated with one-stage/two-stage discrepancies. In this patient the diagnosis of haemophilia A would be missed if only the one-stage assay was used.

von Willebrand's disease (VWD) is an inherited bleeding disorder characterized by deficient levels of or dysfunctional von Willebrand factor (VWF). This phase II/III open-label, multicentre study evaluated the efficacy and safety of BIOSTATE, a high purity plasma-derived double-virus inactivated FVIII/VWF concentrate, when used in non-surgical bleeds, surgical procedures and prophylactic therapy in VWD patients for whom desmopressin treatment was deemed ineffective, inadequate or contraindicated. Twenty three patients (7 type 1, 9 type 2 and 7 type 3; 12 male, 11 female), who received FVIII/VWF concentrate as part of their VWD management, were recruited prospectively between December 2004 and May 2007 from eight centres in Australia and New Zealand. BIOSTATE dosing was based on pre-treatment FVIII:C and/or VWF:RCo plasma levels and a predetermined dosing guide. Haemostatic efficacy of BIOSTATE was rated as excellent or good for all major and minor surgery events, long-term prophylaxis, and for four of the six assessable non-surgical bleeding events. Blood transfusions were required by two major surgery patients as well as one patient with a non-surgical bleed. The median overall exposure to BIOSTATE across all groups was 8 days, greater in the prophylactic group (range 53-197) compared with major surgery (3-24), minor surgery (1-8) and non-surgical bleeds (1-10). BIOSTATE was shown to be efficacious and well tolerated when treating patients with VWD. This study also provides important insights into dosing regimens with BIOSTATE and the role of monitoring therapy with FVIII:C and VWF:RCo.