Passive redirection filters minimize red blood cell contamination during neutrophil chemotaxis assays using whole blood.

Abstract

Neutrophils are the most numerous white blood cells and are the first to arrive at sites of inflammation and infection. Thus, neutrophil behavior provides a comprehensive biomarker for antimicrobial defenses. Several microfluidic tools have been developed to test neutrophil chemotaxis, phagocytosis, extrusion of extracellular traps, etc. Traditional tools rely on purified neutrophil samples, which require lengthy and expensive isolation procedures from large volumes of blood. In the absence of such isolation, visualizing neutrophils in blood is complicated by the overwhelming number of red blood cells (RBCs), which outnumber neutrophils by 1000 : 1. Recently, several microfluidic technologies have been designed to analyze neutrophils directly in blood, by separating neutrophils on selectin coated surfaces before the migration assay or blocking the advance of RBCs with the moving neutrophils. However, RBC contamination remains an issue, albeit with a reduced ratio, down to 1 : 1. Here, we present an RBC-debulking strategy for neutrophil assays based on microscale passive redirection filters (PRFs) that reduce RBC contamination down to as few as a 1 : 17 RBC to neutrophil ratio. We compare the performance of different PRF designs and measure changes in neutrophil chemotaxis velocity and directionality following immune stimulation of whole blood.