Oral Presentation Australasian Cytometry Society 44th Annual Conference and Workshop

Imaging flow cytometry of neutrophil extracellular traps (NETs) (24533)

Patrick M Lelliott 1 , Masayuki Nishide 1 , Nicholas I Smith 1
  1. Osaka university, Osaka, OSAKA, Japan

Neutrophil extracellular traps (NETs) occur when neutrophils mobilize their DNA as a defense mechanism against pathogens. They can release a portion of their DNA, maintaining their cell membrane integrity, known as vital NET formation, or they can self-destruct, unravelling and releasing their entire DNA content as a large antimicrobial cloud.

The analysis of NETs by cytometry has garnered much interest, but presents several unique challenges. The delicate nature, large size, and inherent stickiness of NETs necessitates a unique preparation and analysis procedure. They cannot be centrifuged or filtered, are prone to form large clumps or strings of DNA which can block apertures typical of conventional cytometers, and often produce little FSC/SSC signal due to their diffuse nature.

We have developed a unique preparation and analysis procedure of NETs using imaging flow cytometry1. Applications for this include the analysis of NETs during malaria1, influenza2, vasculitis3, and revealing the unique ability of the commonly used anti-coagulant, heparin, to induce vital NETs4. We will discuss each of these, as well as some of the advantages and disadvantages of cytometry analysis of NETs, with particular emphasis on exploring the role of adhesion and the ability to assess the presence of NETs in blood samples. We will also discuss potential issues with alternate published methods of NET cytometry. To extend the utility of this approach we are developing deep learning models for automated classification of NETs based on imaging cytometry data, and machine learning models based on Raman spectrographic information. Finally, we are applying this technique to investigate the role of microplastics in NET formation.

  1. Lelliott PM, Momota M, Lee MSJ, Kuroda E, Iijima N, Ishii KJ, et al. Rapid Quantification of NETs In Vitro and in Whole Blood Samples by Imaging Flow Cytometry. Cytometry A. 2019 May;95(5):565–78.
  2. Momota M, Lelliott P, Kubo A, Kusakabe T, Kobiyama K, Kuroda E, et al. ZBP1 governs the inflammasome-independent IL-1α and neutrophil inflammation that play a dual role in anti-influenza virus immunity. Int Immunol. 2020 Mar 7;32(3):203–12.
  3. Lelliott PM, Nishide M, Pavillon N, Okita Y, Shibahara T, Mizuno Y, et al. Cellular Adhesion Is a Controlling Factor in Neutrophil Extracellular Trap Formation Induced by Anti-Neutrophil Cytoplasmic Antibodies. ImmunoHorizons. 2022 Feb 1;6(2):170–83.
  4. Lelliott PM, Momota M, Shibahara T, Lee MSJ, Smith NI, Ishii KJ, et al. Heparin induces neutrophil elastase-dependent vital and lytic NET formation. Int Immunol. 2020 May 8;32(5):359–68.