120 From the available questionnaires, 23/81 OI patients with the highest self-BAT bleeding score were selected and invited by telephone for laboratory testing. For these additional blood tests, a separate consent was requested. For comparison purposes, a random sample was taken of 19/114 patients with a score within the normal range of the self-BAT questionnaire. Exclusion criteria were: known pre-existing bleeding diseases, renal or liver diseases, and use of medication that could interfere with haemostasis. We also excluded relatives. Since our centre has a focus on adult OI patients, we included only adult patients ≥ 18 years. Medication use (especially use of anticoagulants) was reaffirmed, as well as liver- and renal dysfunction. Evaluation of bleeding time The IVY method was done as additional test to assess platelet function in combination with possible vessel wall abnormalities 18. The bleeding time test was purposely determined by the same researcher specially trained in the procedure. A fully automatic instrument (Surgicutt adult; depth: 1.0 mm and length: 5.0 mm) was used to determine the bleeding time. Furthermore, a blood pressure cuff (Speidel & Keller Stabil 3), filter paper to staunch the wound, and a timer were used. An 5mm incision was made on the volar side of the forearm lengthwise, approximately 6 cm below the elbow crease. Time was ended when there was no further bleeding after blotting. After 10 minutes of bleeding, the procedure was stopped due to a prolonged bleeding time 18. Blood sampling and laboratory methods Blood was collected through venepuncture of the cubital vein in Vacutainer blood collection tubes (Becton Dickinson; Vianen, the Netherlands) containing 0.109M, 3.2% trisodium citrate or dipotassium ethylene diamine tetraacetic acid (K2EDTA) as anticoagulant. All tests have been standardised using defined reference ranges as criteria for pathological results. The laboratory is subject to national and international external quality assessment in the field of haemostasis and thrombosis. A full blood count was performed on an automated modular haematology system (Sysmex XN9000; Sysmex Europe, Etten-Leur, the Netherlands) for determining haematocrit and platelet count. Activated partial thromboplastin time (APTT), fibrinogen, FVIII activity and von Willebrand factor (VWF) antigen and activity were determined on a Sysmex CS-2500 automated analyser based on turbidimetry. Platelet function was tested on an automated platelet function analyser (PFA-200; Siemens Healthcare Nederland B.V. Den Haag, the Netherlands). It measures the ability of platelets to adhere and aggregate under high shear stress to a membrane covered with a collagen and epinephrine or collagen and ADP 10.
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