Blood was collected 24 h before surgery on sodium citrate from 24 patients (male, aged 69.6 ± 8.7 years, range 60–82) with degenerative AAA,20 (link) and 24 healthy male controls (aged 68.7 ± 8.5 years, range 58–78) examined at ‘Centre d’Investigation Preventative et Clinique’ (IPC, Paris).23 (link) A significant ILT was present in all the AAA studied. Circulating aortic channels were measured at the level of maximal dilatation on CT scan. ILT proportion was calculated as followed: (maximal aortic diameter−circulating channel)/maximal aortic diameter and was expressed as percentage. The mean maximal AAA diameter was 54.0 ± 8.55 mm. The mean ILT thickness was 25.5 ± 8.4 mm, representing 46.8 ± 11.8% of the AAA dilatation.
Samples of AAA thrombus and residual wall were obtained during AAA surgical repair from these 24 and five additional patients. ILT (n = 29) were dissected into luminal, intermediate, and abluminal layers9 (link),20 (link) and the wall was separated into media and adventitia (n = 10). The thrombus layers, media, and adventitia were cut into small pieces (1 mm3) and separately incubated in RPMI-1640 medium (Gibco) for 24 h at 37°C (2 ml/g wet tissue). The tissue-culture media containing released material were then collected and stored at −80°C after determining the protein concentration by the Bradford assay (Bio-Rad). Ethics Committee advice and patient and healthy volunteer informed consent were obtained (RESAA and AMETHYST studies, CPP Paris-Cochin no 2095, 1930, and 1931). The investigation conforms with the principles outlined in the Declaration of Helsinki.
In vitro serum from three healthy volunteers was obtained 2 h and 1, 2, 3, 4, and 7 days after clotting of blood at 37°C. Paired citrated plasma samples were used directly or recalcified (3×10−2 M CaCl2) to induce fibrin formation in the absence of blood cells. Sera, plasma, and recalcified plasma were then kept at −80°C until ELISAs were performed.
Samples of AAA thrombus and residual wall were obtained during AAA surgical repair from these 24 and five additional patients. ILT (n = 29) were dissected into luminal, intermediate, and abluminal layers9 (link),20 (link) and the wall was separated into media and adventitia (n = 10). The thrombus layers, media, and adventitia were cut into small pieces (1 mm3) and separately incubated in RPMI-1640 medium (Gibco) for 24 h at 37°C (2 ml/g wet tissue). The tissue-culture media containing released material were then collected and stored at −80°C after determining the protein concentration by the Bradford assay (Bio-Rad). Ethics Committee advice and patient and healthy volunteer informed consent were obtained (RESAA and AMETHYST studies, CPP Paris-Cochin no 2095, 1930, and 1931). The investigation conforms with the principles outlined in the Declaration of Helsinki.
In vitro serum from three healthy volunteers was obtained 2 h and 1, 2, 3, 4, and 7 days after clotting of blood at 37°C. Paired citrated plasma samples were used directly or recalcified (3×10−2 M CaCl2) to induce fibrin formation in the absence of blood cells. Sera, plasma, and recalcified plasma were then kept at −80°C until ELISAs were performed.
