Flow cytometry detection of Shiga toxins in the blood from children with hemolytic uremic syndrome

Background: Hemolytic uremic syndrome (HUS) is the main cause of acute renal failure in early childhood. Most cases are due to intestinal infections from Escherichia coli strains (STEC) which produce Shiga toxins (Stxs). Stx1 and Stx2 produced by STEC in the gut are absorbed into the circulation and, after binding on polymorphonuclear leukocytes (PMNs), are targeted to renal endothelium. The aim of the present work was the development of a method to detect Stxs bound on circulating PMN and diagnose STEC infections in patients with HUS. Methods: White blood cells isolated after erythrocytes lysis were incubated with anti-Stxs mouse monoclonal antibodies in the presence of human serum to saturate Fc receptors on PMNs. After incubation with FITC-goat anti-mouse IgG, flow cytometry analysis was utilized to reveal the cell-bound fluorescence. Results: The method was quick (3 h), sensitive (femtomoles) and capable to detect both Stxs. The presence of Stxs was detected on PMNs from six patients with HUS: four patients had serological or microbiological evidence of STEC infection, whereas the other two patients had no evidence of STEC infection when employing the standard diagnostic methods. Conclusions: The method described is rapid, simple, based on commercially available reagents and it might be more sensitive than the standard methods for diagnosis of STEC infection. It also allows the detection of Stxs in blood, a key step to monitor the pathogenesis of HUS.