Calibrated Automated Thrombography Sample Clauses

Calibrated Automated Thrombography. The Thrombinoscope TM assay (Thrombinoscope BV, maastrichts, Netherlands) was used to measure thrombin generation, as described by Xxxxxx et al, 2003. Fresh platelet poor plasma was produced by double centrifugation at 4750 g for 10 min on both occasions and decanting the top three-quarter supernatant after each centrifugation, initially into a polypropylene tube then finally into a plastic tube suitable for freezing at - 40˚C. The PPP was frozen at -40˚C within 1h of venepuncture. PPP was thawed in 37˚C waterbath immediately prior to analysis, within one month of collection. The reagents used were PPP-Reagent Low and FluCa-kit, together which contain 1 pM tissue factor and 4 µM phospholipids. FluCa-kit consists of Fluo-Buffer, containing calcium chloride, and Fluo-Substrate, containing a fluorogenic substrate. 20 ul of the PPP-Reagent Low was added to test xxxxx 1-3 of the immulon 96-well micro titre plate (Thermo Labsystems, Franklin, USA). The calibration xxxxx were run in triplicate in which 20 ul of thrombin calibrator was placed into xxxxx 4-6 of the same row. 80 ul of thawed patient PPP was added to each well of that patient nominated row. The PPP with CTI were treated as separate patient samples and run in separate rows to the PPP without CTI. The plate was placed inside the CAT and incubated for 10 minutes. The fluo-buffer was warmed and 40 ul fluo-substrate was added and placed on the vortex. 20 ul of the mixed fluo-reagent was dispensed automatically by the fluorometer, into each well, which automatically triggered coagulation. Fluoroscan Ascent measured the fluorescence over time. The filters in place were 390 nm excitation and 460 nm emission. The Thrombinoscope BV software (version 3.0.0.30) converted the signal into nM thrombin generated over time and adjusted both for the filter and the thrombin bound to alpha 2 macroglobuin. Controls using reconstituted PPP were run with each plate. Non-human control plasma, NHCP (Technoclone, Vienna, Austria), was used for both intra- and inter-assay variability assessments. NHCP was reconstituted as per manufacturer’s instructions, pooled and frozen to -40C before use. Intra-assay variability was assessed using NHCP in one plate of 16 assays and each assay was run in triplicate. Inter-assay variability was assessed using pooled, frozen, human control plasma and NHCP, run in eight triplicate assays each.