Development and validation of a new assay for assessing clot integrity |
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| Affiliation: | 1. Center for Reproductive Medicine, Academic Medical Center, Amsterdam, The Netherlands;2. Department of Obstetrics and Gynecology, Radboud University Medical Center, Nijmegen, The Netherlands;3. Center for Reproductive Medicine, UZ Brussel, Free University of Brussels, Belgium;4. Department of Reproductive Medicine, University Medical Center Utrecht, Utrecht, The Netherlands;5. Department of Reproductive Medicine, VU University Medical Center, Amsterdam, The Netherlands;6. Department of Reproductive Medicine, Maastricht University Medical Center, Maastricht, The Netherlands;7. Department of Obstetrics and Gynaecology, St Elisabeth Hospital, Tilburg, The Netherlands;8. Department of Gynaecology, Leiden University Medical Centre, Leiden, The Netherlands;9. Department of Obstetrics and Gynaecology, University Medical Center Groningen, Groningen, The Netherlands;10. Department of Obstetrics and Gynecology, Erasmus Medical Center, Rotterdam, The Netherlands;11. Department of Obstetrics and Gynecology, Jeroen Bosch Hospital, Den Bosch, The Netherlands;12. Assisted Conception Unit, Guy''s and St Thomas'' Hospital NHS Foundation Trust, London, United Kingdom;13. Department of Medical Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands;1. Matrix Biology Program, Benaroya Research Institute, 1201 Ninth Avenue, Seattle, WA 98101, USA;2. Department of Pathology, University of Washington, Seattle, WA 98195, USA;3. Department of Biochemistry, University of Texas Health Science Center at Tyler, Tyler, TX 75708, USA |
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| Abstract: | IntroductionResearch and routine laboratory assessment of clot integrity can be time consuming, expensive, and cannot be batched as it is generally performed in real time. To address these issues, we developed and validated a micro-titre based assay to quantify thrombogenesis and fibrinolysis, the purpose being to assess patients at risk of cardiovascular events by virtue of hypercoagulability. In further validation, thrombogenesis results were compared to similar indices from the thrombelastograph (TEG).MethodsOur assay determines three indices of thrombogenesis (lag time to the start of thrombus formation (LT), rate of clot formation (RCF), and maximum clot density (MCD)) and two of fibrinolysis (rate of clot dissolution (RCD) and time for 50% of the clot to lyse (T50)). Plasma was tested fresh and again after being frozen at − 70 °C. Some samples were tested immediately, others after being left at room temperature for up to 24 h.ResultsThe intra-assay coefficients of variation (CVs) of the three thrombogenesis measures (LT, RCF, MCD) and two fibrinolysis measures (RCD, T50) varied between 2.7 and 12.0% in fresh plasma and between 1.3% and 10.8% in frozen plasma respectively. Similarly, the inter-assay coefficients of variation of the thrombogenesis and fibrinolysis measures were 4.9–10.8% in fresh plasma and 2.2–6.5% in frozen plasma respectively. TEG assays intra- and inter assay CVs were around 25%. There were no significant differences in all plate assay indices up to 6 h at room temperature. Certain plate assay thrombogenesis data were comparable to TEG indices after analysis by Pearson's correlation. The reagent processing cost per sample is £15 for TEG and £2 for the plate assays.ConclusionOur micro-titre based assay assessing plasma thrombogenesis and fibrinolysis has good intra- and inter-assay CVs, can assess plasma up to 6 h after venepuncture, is more efficient (in terms of throughput) and is more economical than that of the TEG. |
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