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. 2023 Jun 20;13(12):e4701.
doi: 10.21769/BioProtoc.4701.

A New Methodology for the Quantification of Neutrophil Extracellular Traps in Patient Plasma

Bharati Matta  1 , Jenna Battaglia  1 , Betsy J Barnes  1   2
Affiliations

A New Methodology for the Quantification of Neutrophil Extracellular Traps in Patient Plasma

Bharati Matta et al. Bio Protoc. .

Abstract

Neutrophil extracellular traps (NETs) are web-like structures made up of decondensed chromatin fibers along with neutrophil granular proteins that are extruded by neutrophils after activation or in response to foreign microorganisms. NETs have been associated with autoimmune and inflammatory diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, coronavirus disease 2019 (COVID-19), and others. There are reliable methods available to quantitate NETs from neutrophils, but their accurate quantification in patient plasma or serum remains a challenge. We developed a highly sensitive ELISA to detect NETs in serum/plasma and designed a novel smear immunofluorescence assay to detect NETs in as little as 1 μL of serum/plasma. We further validated our technology on plasma samples from SLE patients and healthy donors that carry interferon regulatory factor 5 genetic risk. The multiplex ELISA combines the use of three antibodies against myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and DNA to detect the NET complexes with higher specificities. The immunofluorescence smear assay can visually detect intact structures of NETs in 1 μL of serum/plasma and provide similar results that correlate with findings from the multiplex ELISA. Furthermore, the smear assay is a relatively simple, inexpensive, and quantifiable method of NET detection for small volumes.

Keywords: ELISA; Immunofluorescence; NETosis; Quantification; Smear assay.

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Conflict of interest statement

Competing interestsThe authors declare no conflicts of interest.

References

    1. Bendib I., de Chaisemartin L., Granger V., Schlemmer F., Maitre B., Hue S., Surenaud M., Beldi-Ferchiou A., Carteaux G., Razazi K., et al. .(2019). Neutrophil Extracellular Traps Are Elevated in Patients with Pneumonia-related Acute Respiratory Distress Syndrome. Anesthesiology 130(4): 581-591. - PubMed
    1. Bruschi M., Bonanni A., Petretto A., Vaglio A., Pratesi F., Santucci L., Migliorini P., Bertelli R., Galetti M., Belletti S., et al. .(2020). Neutrophil Extracellular Traps Profiles in Patients with Incident Systemic Lupus Erythematosus and Lupus Nephritis. J Rheumatol 47(3): 377-386. - PMC - PubMed
    1. Corsiero E., Pratesi F., Prediletto E., Bombardieri M., and Migliorini P.(2016). NETosis as source of autoantigens in rheumatoid arthritis. Front immunol 7: 485. - PMC - PubMed
    1. Garcia-Romo G. S., Caielli S., Vega B., Connolly J., Allantaz F., Xu Z., Punaro M., Baisch J., Guiducci C., Coffman R. L., et al. .(2011). Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med 3(73): 73ra20. - PMC - PubMed
    1. Kaplan M. J. and Radic M.(2012). Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol 189(6): 2689-2695. - PMC - PubMed