Haemodilution-induced changes in coagulation and effects of haemostatic components under flow conditions†  S. Ogawa, T. Ohnishi, K. Hosokawa, F. Szlam,

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Haemodilution-induced changes in coagulation and effects of haemostatic components under flow conditions†  S. Ogawa, T. Ohnishi, K. Hosokawa, F. Szlam, E.P. Chen, K.A. Tanaka  British Journal of Anaesthesia  Volume 111, Issue 6, Pages 1013-1023 (December 2013) DOI: 10.1093/bja/aet229 Copyright © 2013 The Author(s) Terms and Conditions

Fig 1 Schemata of the flow-chamber system. Total Thrombus Formation Analysis System (T-TAS). British Journal of Anaesthesia 2013 111, 1013-1023DOI: (10.1093/bja/aet229) Copyright © 2013 The Author(s) Terms and Conditions

Fig 2 Video microscopic images of thrombus formation under flow conditions. (a) During perfusion at 10 µl min−1, small thrombus was deposited on the capillary within 3–4 min. Rapid growth of thrombus was observed, occupying the capillary just 1–2 min after the appearance of thrombus. (b) The initial appearance of thrombus at 3 µl min−1 was similar to perfusion at 10 µl min−1, but subsequent growth of thrombus was slower at 3 µl min−1. *Occlusion occurred in the capillary. After 40% dilution of whole blood, the initial appearance of thrombus was prolonged for perfusion at 10 µl min−1, while it was not affected at 3 µl min−1. Conversely, subsequent growth of thrombus was delayed at 3 µl min−1 more than at 10 µl min−1. Addition of vWF to diluted whole blood accelerated appearance of thrombus at 10 µl min−1. Adding PCC increased both intra-luminal and mural thrombus formation at both flow rates. Addition of fibrinogen (Fgn) concentrate increased the thickness of mural thrombus, but did not cause the complete capillary occlusion. British Journal of Anaesthesia 2013 111, 1013-1023DOI: (10.1093/bja/aet229) Copyright © 2013 The Author(s) Terms and Conditions

Fig 3 Analysis of thrombus by confocal laser scanning microscopy. Blood was perfused from left to right for all panels. For each panel, fibrin stained by anti-Fibrinogen-Alexa594 (left panel), platelets stained by anti-CD41-FITC (middle panel), and merged platelet and fibrin staining (right panel) are shown. The white bar represents 100 µm. The thrombus formed in the mid-segment of the microchip capillary was composed of platelets (in green) containing fibrin strands (in red). Fgn, fibrinogen. British Journal of Anaesthesia 2013 111, 1013-1023DOI: (10.1093/bja/aet229) Copyright © 2013 The Author(s) Terms and Conditions

Fig 4 Immunoblot analysis of thrombus. (a) Thrombin detection: in control blood, thrombus contained considerable amounts of thrombin at both 10 and 3 µl min−1. After 40% dilution, decreased amounts of thrombin were observed at both flow rates. The addition of PCC to diluted blood increased the amount of thrombin in the thrombus at both flow rates, while there were no significant changes after supplementing vWF. (b) Fibrin detection: there was more abundant thrombin-catalysed fibrin species (α-, β-, γ-chains and γ-dimers) in the thrombus formed at 3 compared with 10 µl min−1. After dilution, these fibrin chains were decreased at both flow rates. The decreases in fibrin after dilution were reversible with fibrinogen replacement at 3 µl min−1. The combination of PCC and fibrinogen increased both thrombin and fibrinogen content at both flow rates. Fgn, fibrinogen. British Journal of Anaesthesia 2013 111, 1013-1023DOI: (10.1093/bja/aet229) Copyright © 2013 The Author(s) Terms and Conditions

Fig 5 Effects of haemodilution and haemostatic components in whole blood from. (a) Representative flow pressure patterns. At 10 µl min−1, onset of thrombus formation (Ton) was more affected than capillary occlusion (T80) by haemodilution and corrective haemostatic interventions. On the other hand, T80 was more affected than Ton at 3 µl min−1. (b) Onset of thrombus formation (Ton), and (c) time to capillary occlusion (T80). After 40% dilution, both Ton and T80 were prolonged at both flow rates. Addition of vWF shortened Ton at 10 µl min−1, but not at 3 µl min−1. PCC shortened T80 at 3 µl min−1. However, addition of fibrinogen to diluted whole blood prolonged Ton at 10 µl min−1 and T80 at both flow rates. After addition of both fibrinogen and PCC, Ton at 3 µl min−1 was shortened by 46% compared with Ton after 40% dilution. *P<0.05 vs control; **P<0.05 vs 40% dilution. Fgn, fibrinogen. British Journal of Anaesthesia 2013 111, 1013-1023DOI: (10.1093/bja/aet229) Copyright © 2013 The Author(s) Terms and Conditions

Fig 6 Effects of haemodilution and haemostatic components in whole blood after CPB. Onset of thrombus formation (Ton) and the capillary occlusion (T80) after CPB. Addition of PCC shortened both Ton and T80. In contrast, addition of fibrinogen prolonged both parameters. The combination of PCC and fibrinogen shortened both Ton and T80. *P<0.05 vs baseline; **P<0.05 vs post-CPB. Fgn, fibrinogen. British Journal of Anaesthesia 2013 111, 1013-1023DOI: (10.1093/bja/aet229) Copyright © 2013 The Author(s) Terms and Conditions