Microfluidics to study deformability of human erythrocytes exposed to alpha hemolysin.

LEAL DENIS, MARÍA FLORENCIA; OSTUNI, MARIANO ANIBAL; EL NEMER, WASSIM; LIZARRALDDE IRAGORRI, MARÍA ALEJANDRA; SCHWARZBAUM, PABLO JULIO

Rosario

Congreso; Sociedad Argentina de Fisiologíaa. Reunión Anual 2019.; 2019

Sociedad Argentina de Fisiología

The hemolytic toxin alpha-hemolysin (HlyA) is a virulence factor produced by several strains of Escherichia coli. It is involved in urinary tract infections, peritonitis, meningitis and septicemia. HlyA of E. coli irreversibly binds to human erythrocytes (RBCs) initiating a series of biochemical and morphological events leading to hemolysis.We studied the capacity of HlyA-treated RBCs to deform under mechanical stress, by passing the cells though amicrofluidic device (biochip) that combines a network of microchannels of 5 um high and a serial of Restrictions of 5 μm separated by 20 and 30 μm-wide areas, designed to mimicked the mechanical stress exerted on RBCs in the microcirculation.RBCs (20% hematocrit) were treated with 0.1-0.2 ng/uL of HlyA (HlyA-RBCs), with 1-2 ng/uL of the un-acylatedprotoxin ProHlyA (Pro-RBCs) or vehicle (c-RBCs) and loaded on the biochip. The biochip was connected to apump (350 mBar) and mounted on an inverted microscope coupled with a high speed camera. Samples were perfused through the biochip and frame sequences ware taken throughout the experiment. Hemolysis due to mechanical stress was evaluated by measuring free hemoglobin and deformability was estimated through the deformation index (DI).Results showed no significant differences in the hemolytic response among different treatments and thecontrol (0.3 ?1.0 %). On the other hand, exposure to 0.2 and 2 ng/uL ProHlyA reduced 16 % the DI, while exposure to HlyA 0.1 and 0.2 ng/uL reduced deformability 30 % and 35 %, respectively.HlyA-treated RBCs were equally resistant to lysis than cRBCs and ProHlyA-RBCs in the experimental conditions.However, both Pro- and HlyA-treated RBCs presented a lower DI, indicating loss of cell elasticity. In Pro-RBCs the DI was independent of the assayed concentration since no further decreased was observed with higherconcentrations of ProHlyA. On the other hand, exposure to increased concentrations of HlyA leaded to a slight but significant decrease on the DI. It can be concluded that in the assayed conditions, all treated-RBCs showed sufficient flexibility to allow them to bend in order to flow through narrow capillaries without hemolysing.