Summarymedium may produce false positive results (11). In this report, we describe two modifications of the original Bethesda assay which result Antibodies against factor VIII coagulant activity can appear in in markedly increased specificity and allowed us to diagnose and monihaemophiliacs who are treated with factor VIII preparations but also tor the presence of factor VIII :C inhibitors, spontaneously in non-haemophiliacs. The Bethesda assay is the most commonly used method to detect these antibodies, but it lacks specificity especially in the lower range resulting in unreliable data.Two modifications are proposed and tested to resolve the imper fections: 1. Buffering the normal plasma used in the assay-and control mixture with 0.1 M imidazole to pH 7.4. p 2. Replacing the imidazole buffer in the control mixture by immunodepleted factor VIII deficient plasma. These modifications allow better discrimination between positive and negative samples and improve reliability.

This study is the first in a series to assess the psychometric properties of the HJHS, a promising new measure of joint health in boys with haemophilia.

To prevent hemophilic arthropathy, prophylactic treatment of children with severe hemophilia should be started before joint damage has occurred. However, treatment is expensive, and the burden of regular venipunctures in young children is high. With the aim of providing information on starting prophylaxis on the basis of individual patient characteristics, the effect of postponing prophylaxis on longterm arthropathy was studied in a cohort of 76 patients with severe hemophilia born between 1965 and 1985. The median age at first joint bleed was 2.2 years (range, 0.2-5.8). Prophylaxis was started at a median age of 6 years (interquartile range [IQR], 4-9), and the median annual clotting factor use on prophylaxis was 1750 IU/kg/y (31 IU/kg/wk). Hemophilic arthropathy was measured by the Pettersson score (maximum, 78 points). At a median age of 19 years, the median Pettersson score was 7 points (IQR, 0-17). After 2 decades of follow-up, the Pettersson score was 8% higher (95% confidence interval, 1%-16%) for every year prophylaxis was postponed after the first joint bleed. This effect was independent of age at Pettersson score, age at first joint bleed, and prophylactic dose used. In conclusion, most patients have their first joint bleed after the age of 2 years. Patients who start prophylaxis soon after the first joint bleed show little arthropathy in adulthood. The longer the start of prophylaxis is postponed after the first joint bleed, the higher the risk of developing arthropathy.

Studies have shown that joint bleeding leads to cartilage degradation independent of concurrent synovitis. We hypothesized that the blood-induced cartilage damage is because of increased chondrocyte apoptosis after shortterm exposure of whole blood or isolated mononuclear cells plus red blood cells to cartilage. Human cartilage tissue samples were co-cultured for 4 days with whole blood (50% v/v) or with mononuclear cells plus red blood cells (50% v/v equivalents). Cartilage matrix proteoglycan synthesis ( 35 SO 4 2؊ incorporation) was determined after 4 days as well as at day 16 (after a 12-day recovery period in the absence of any additions). To test the involvement of apoptosis a specific caspase-3 inhibitor (acDEVDcho, 0 to 500 mol/L) as well as a pancaspase inhibitor (zVADfmk, 0 to 500 mol/L) were added. Chondrocyte apoptosis was evaluated by immunohistochemical staining of single-strand DNA and by terminal dUTP nick-end labeling. Cartilage co-cultured with whole blood as well as mononuclear cells plus red blood cells induced a long-term inhibition of proteoglycan synthesis (74% and 78% inhibition on day 16, respectively). Immunohistochemistry showed a threefold increase in apoptotic chondrocytes in cultures with 50% whole blood as well as with mononuclear cells plus red blood cells. Both the specific caspase-3 inhibitor and the pan-caspase inhibitor partially restored proteoglycan synthesis in the cartilage after blood exposure. This effect was accompanied by a decrease in the number of apoptotic chondrocytes. These data suggest that a single joint hemorrhage (a 4-day exposure of cartilage to 50% v/v blood) results in induction of chondrocyte apoptosis, responsible for the observed inability of the chondrocytes to restore the proteoglycan synthesis during recovery from a short-term exposure to blood. This reduced restoration could eventually lead to cartilage degeneration and ultimately joint destruction.

SummaryTAFI (thrombin activatable fibrinolysis inhibitor) down regulates fibrinolysis after activation by relatively high concentrations of thrombin generated during coagulation via thrombin mediated factor XI activation and subsequent activation of the intrinsic pathway. It is this secondary burst of thrombin that is severely diminished in haemophilia A, a deficiency of coagulation factor VIII. We therefore investigated the role of TAFI in haemophilia A by measuring the clot lysis times of tissue factor induced fibrin formation and tPA mediated fibrinolysis. In haemophilia A plasma clot lysis times were normal at relatively high tissue factor concentrations but severely decreased at moderate to low tissue factor concentrations, indicating that the thrombin generation via the extrinsic pathway was insufficient to activate TAFI. Addition of factor VIII, TAFI or thrombomodulin restored the clot lysis times at low tissue factor concentrations. This confirms the hypothesis that the bleeding disorder in haemophilia A is not merely a defect in the initial clot formation but is in fact a triple defect: reduced thrombin formation via the extrinsic pathway at low tissue factor concentrations, a reduced secondary burst of thrombin generation via the intrinsic pathway and a defective down regulation of the fibrinolytic system by the intrinsic pathway.

In a multinational working group, an instrument (Haemo-QoL) to assess quality of life in children/adolescents with haemophilia and their parents has been developed. In co-operation with haemophilia treatment centres in six European countries, approximately 10 children/adolescents with haemophilia per country and their parents were asked to participate in the pilot-testing. Both self-reported and parent-reported questionnaires were provided for two age-groups of children (4-16 years). Medical data was collected from physicians from patient files. Answers to open questions from participants (58 children and 57 parents) confirmed the content of 116 of the preliminary items. Cognitive debriefing revealed that the majority of the Haemo-QoL was rated favourably, but 29 questions were recommended to be omitted and several items to be reformulated. Preliminary psychometric testing of the revised 77 item questionnaire in the same sample showed acceptable reliability and validity, which will be examined in a subsequent study with a larger patient sample.

The incidence of FIX inhibitors in severe hemophilia B (SHB) is not well defined. Frequencies of 3-5% have been reported but most studies to date were small, including patients with different severities, and without prospective follow-up for inhibitor incidence. Study objective was to investigate inhibitor incidence in patients with SHB followed up to 500 exposure days (ED), the frequency of allergic reactions, and the relationship with genotypes. Consecutive previously untreated patients (PUPs) with SHB enrolled into the PedNet cohort were included. Detailed data was collected for the first 50 ED, followed by annual collection of inhibitor status and allergic reactions. Presence of inhibitors was defined by at least two consecutive positive samples. Additionally, data on factor IX gene mutation was collected. 154 PUPs with SHB were included; 75% were followed until 75 ED, and 43% until 500 ED. Inhibitors developed in 14 patients (7 high-titre). Median number of ED at inhibitor manifestation was 11 (IQR 6.5-36.5). Cumulative inhibitor incidence was 9.3% (95%CI 4.4-14.1) at 75 ED, and 10.2% (5.1-15.3) at 500 ED. Allergic reactions occurred in 4 (28.6%) inhibitor patients. Missense mutations were most frequent (46.8%) overall but not associated with inhibitors. Nonsense mutations and deletions with large structural changes comprised all mutations among inhibitor patients and were associated with an inhibitor risk of 26.9% and 33.3%, respectively. In an unselected, well-defined cohort of PUPs with SHB, cumulative inhibitor incidence was 10.2% at 500 ED. Nonsense mutations and large deletions were strongly associated with the risk of inhibitor development. The PedNet Registry is registered at clinicaltrials.gov; identifier: NCT02979119

A survey was made of the current status of treatment of haemophilic boys at 20 centres in 16 European countries and includes approximately 1500 of the estimated 6500 haemophiliacs in the participating countries. Many mild haemophiliacs are not seen, or seen infrequently, at haemophilia centres and this requires study. Nine of 18 centres provide continuous prophylaxis to 80-100% of their patients, five centres provide it to 55-80% and the remaining four centres to 15-40% of the boys. The median dose given was 6240 U kg-1 year-1 (range 3120-7800). Four centres administered only recombinant concentrates to children with severe haemophilia A, while seven centres administered recombinant concentrates to 75-90% and the remaining centres to less than 50% of the boys (two centres < 10%). When asked for the choice of concentrate for a newly diagnosed boy with severe haemophilia A, all but one centre preferred recombinant concentrate. Most boys below 6 years received concentrates via a peripheral vein but three centres preferred a central venous line for 80-100% of the boys. Thirteen of 18 centres applied home treatment to 84-100% of the boys and the remaining five centres to 57-77% of the boys.