All laboratory tests except FBC will be conducted in the Haemostasis & Thrombosis Laboratories at Guy’s & St Thomas’ Hospital. Dade® Innovin® recombinant thromboplastin, Actin FS®, Thromboclotin®, Thrombin-Reagent®, Innovance D-dimer and HYPHEN BioMed Biophen FVIII:C chromogenic assay (Siemens Healthcare, Marburg, Germany) will be used on Sysmex CS2100i coagulation analysers (Sysmex UK, Milton Keynes, UK) for PT, APTT, Clauss fibrinogen, D-dimers and FVIII activity respectively. For lupus anticoagulant detection, DRVVT will employ Life Diagnostics LA Screen and LA Confirm reagents (Diagnostica Stago UK, Theale, UK), and DAPTT will use Stago PTT-LA (Diagnostica Stago) in the screen and addition of Bio/Data Corporation LA Confirmation Reagent (Alpha Labs, Eastleigh, UK) for the confirm. LA assays will be performed on a Sysmex 2000i analyser (Sysmex UK). Chromogenix Coamatic antithrombin, Coamatic protein C, Coatest APC Resistance, Coatest APCR resistance-V (Quadratech Diagnostic Ltd., Epsom, UK) and STA®-Liatest® Free protein S (Diagnostica Stago) will be used on Sysmex CS2000i analysers for antithrombin activity, protein C activity, phenotypic activated protein C resistance and free protein S antigen respectively. Inova Diagnostics QUANTA Lite® (ELISA) kits for IgG and IgM anticardiolipin and anti-β
2 glycoprotein I antibodies (Werfen UK, Warrington, UK), Enzygnost® F1 + 2 and TAT will be performed on a Dynex DS2 ELISA analyser (Werfen UK) for anticardiolipin antibodies, anti-β
2 glycoprotein I antibodies, prothrombin fragment 1 + 2 and thrombin-antithrombin complexes respectively. HemosIL VWF Antigen will be performed on an ACL TOP 500 coagulation analyser (Werfen UK) for von Willebrand factor antigen. Platelet function screening will be performed on a PFA-100 analyser (Sysmex UK) with collagen/ADP and collagen/epinephrine cartridges (Sysmex UK). Homocysteine will be assayed with Abbott ARCHITECT chemiluminescent microparticle assay (Abbott UK, Maidenhead, UK) on an Abbott ARCHITECT
i2000SR immunoassay analyser (Abbott UK). The Thrombin Generation Assay will performed using a Technoclone kit (Technoclone, Vienna, Austria) on a Wallac Victor
3 multilabel plate reader (Perkin Elmer, Turku, Finland). The Clot Lysis Assay will be performed as previously described [
6] and read on a Wallac Victor
3 multilabel plate reader (Perkin Elmer, Turku, Finland). The Prothrombin 20,210 polymorphism will be screened for using an in house developed allelic discrimination assay on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).