A MICROFLUIDIC LEUKOCYTE ROLLING AND ADHESION ASSAY FOR RAPID INFECTION DIAGNOSIS USING A FINGERPRICK VOLUME OF BLOOD

Abstract

Sepsis is a severe clinical syndrome triggered by a pathogen invasion into an organism and the resulting subsequent inflammatory response of the body’s immune system to it. For long time now, the state of the art method for infection diagnosis was based on blood culture, turnaround time of which (7 days) considerably delayed clinical decision making and prevented timely administration of the antibiotics. We developed a new method for early sepsis diagnosis from a drop of blood, based on upregulated expression of cell adhesion ligands on leukocytes and their resulting differential capture to the biomimetic microfluidic device. We found that expression of PSGL-1, a primary ligand that enables leukocyte binding to P-selectin and rolling on endothelial bed, is upregulated in response to systemic infection. This finding led us to hypothesize that the higher expression of PSGL-1 on activated leukocytes would result in elevated frequency of leukocyte recruitment and capture on the activated endothelium when perfused with infected blood samples. We developed an inflamed endothelium-mimicking surface by functionalizing the aldehyde-coated glass substrate of the microchannel with a set of the cell adhesion molecules (CAMs) (P-selectin, E-selectin, ICAM-1). We found that when perfused with the infected blood samples, higher percentage of the leukocytes were captured on the biomimetic surface than when perfused with the healthy blood samples. Further validation of our method allowed us to differentiate rats infected with a range of Gram-negative, Gram-positive bacteria and endotoxin from models of sham-infected and healthy rats. Because the on-chip assay we propose is capable of detecting infection as early as 1-hour post-infection and requires as little as 50 μl of blood for analysis, we believe that this platform could significantly benefit the point-of-care diagnostics of sepsis in a portable device.