The detection of circulating endothelial cells in human septic shock

The vascular endothelium has a central role in the control of microvascular tone and it has been hypothesized that vascular endothelial damage occurs in septic shock, producing multi-organ failure. 1 Isolated endothelial cells (EC) exposed to pro-inflammatory cytokines and other mediators upregulate surface adhesion molecules, synthesize vasoactive compounds and express pro-coagulant factors. However, there is little direct evidence of EC damage in human sepsis due to the inaccessibility of the vascular endothelium. Circulating EC have been detected in a number of vascular diseases including coronary artery and sickle cell disease. We therefore hypothesized that circulating EC would be detectable in septic shock. Cell seeding experiments were initially performed to validate the isolation method. Known concentrations of human umbilical endothelial cells (HUVECS) were seeded into whole blood and recovered by an isopycnic centrifugation method using Percoll suspension. EC were identified using antibodies to Factor VIII and the vascular endothelial growth factor receptor, KDR by an indirect immunofluorescence technique using FITC and PE-conjugated secondary antibodies. EC were counted using an inverted fluorescence microscope and expressed as recovered cells per ml of blood. A recovery rate of over 90% was achieved with cell concentration between 50 and 100 cells ml −1 . At a dilution of 10 cells ml −1 recovery fell to less than 40%. Non-specific binding was assessed using isotype control antibodies and subtracted from the final count. Blood samples were taken from freshly placed and flushed venous lines from 11 healthy volunteers, nine ventilated ICU control patients without sepsis and 15 patients with septic shock. The cells were separated, identified and counted as described above. Cell counts using the Factor VIII antibody were consistently higher than those using KDR but correlation between the two counts was high ( r =0.98) (Table 11). There was a significant increase in circulating EC, identified with both Factor VIII and KDR, in patients with septic shock, compared to both healthy volunteers and ICU controls ( P