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 by Shiga toxin (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 PMNs and to diagnose STEC infections in patients with HUS.


METHODS
White blood cells isolated after erythrocytic lysis were incubated with anti-Stxs mouse monoclonal antibodies in the presence of human serum to saturate Fc receptors on PMNs. After incubation with fluorescein isothiocyanate-goat anti-mouse immunoglobulin G, flow cytometric analysis was used to demonstrate the cell-bound fluorescence.


RESULTS
The method was quick (3 h), sensitive (femtomoles), and capable of detecting both Stxs. The presence of Stxs was detected on PMNs from six patients with HUS: four patients had serologic 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, and 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.
Tazzari
Pier Luigi
PL
Servizio di Immunoematologia e Trasfusionale, Ospedale S. Orsola-Malpighi, Bologna, Italy.
Ricci
Francesca
F
Carnicelli
Domenica
D
Caprioli
Alfredo
A
Tozzi
Alberto E
AE
Rizzoni
Gianfranco
G
Conte
Roberto
R
Brigotti
Maurizio
M
D000293:Adolescent; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002648:Child; D002675:Child, Preschool; D004912:Erythrocytes / Q000166:cytology; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006463:Hemolytic-Uremic Syndrome / Q000097:blood* / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007223:Infant; D007231:Infant, Newborn; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D051437:Renal Insufficiency; D012680:Sensitivity and Specificity; D022621:Shiga Toxin / Q000097:blood*; D022622:Shiga Toxin 1 / Q000378:metabolism; D022641:Shiga Toxin 2 / Q000378:metabolism; D013997:Time Factors
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D022621:Shiga Toxin; D016650:Fluorescein-5-isothiocyanate