Measurement of Immune and Endothelial Markers

Whole blood samples were collected in sterile Vacutainer™ (Becton Dickinson, Franklin Lakes, NJ) acid citrate dextrose tubes and processed within 12 h. The PBMC and plasma samples were stored at −180 °C in liquid nitrogen and −80 °C, respectively, in non-pyrogenic polypropylene cryovials (Nunc Cryotubes™) [28 (link)]. LPS levels were quantified using the Limulus Amebocyte Lysate [LAL] chromogenic assay (Lonza Inc., Allendale, NJ) as previously described [28 (link)]. The plasma samples were diluted 1:4 in 0.15 M NaCl prior to analysis, and the lower limit of detection was 0.1 endotoxin units [EU] per milliliter. The following soluble markers of immune and endothelial activation were measured by ELISA: sCD14, osteopontin [OPN], C-reactive protein [CRP], soluble ICAM-1 [sICAM-1], sVCAM-1 (R&D Systems Inc., Minneapolis, MN), and IL-6 (BD Biosciences, San Diego, CA). The coagulation marker D-dimer was measured by ELISA (American Diagnostica GmbH, Stamford, CT).