Background

Neutrophils are the most abundant leukocyte in the human blood and are the first cells to respond to an external stimulus such as a pathogen and/or inflammatory trigger (Kaplan and Radic, 2012; Matta et al., 2022). They migrate to the site of infection where they protect the host by phagocytosing, killing, and digesting pathogens (Kaplan and Radic, 2012; Matta et al., 2022). Another powerful tool neutrophils use to defend the body is the release of neutrophil extracellular traps (NETs). NETs can trap microorganisms and kill them by releasing granular antimicrobial peptides and proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), and cathepsin G(3); however, when this process gets dysregulated or NET clearance is diminished, it can lead to tissue damage, autoimmunity, and other inflammatory conditions (Lande et al., 2011; Kaplan and Radic, 2012; Corsiero et al., 2016; Bruschi et al., 2020; Matta et al., 2022).

Formation of NETs involves the citrullination of histones, chromatin decondensation, and disintegration of the nuclear membrane followed by the release of nuclear content along with proteins such as MPO and NE (Kaplan and Radic, 2012; Matta et al., 2022). Traditionally, NETs are detected by using either anti-MPO or anti-citrullinated histone H3 (CitH3), or neutrophil elastase as coating antibody separately followed by anti-DNA detecting antibody (Zuo et al., 2020 and 2021). We have developed and validated a more sensitive ELISA, where we use a cocktail of anti-MPO and anti-CitH3 antibodies as coating antibodies, followed by anti-DNA as the detecting antibody. We further optimized the best blocking and substrate combination suited for increased sensitivity for ELISA. To the best of our knowledge, we are the first one to develop the smear assay to visualize NETs in small amounts of patient plasma. This method requires minimal time, reagents, specialized equipment, and/or cost. These techniques can be applied to detection of NETs in serum/plasma of patients with autoimmune diseases and other inflammatory conditions where NETs have been shown to play a crucial role. But apart from that, we propose that the application of these techniques can be extended to other bodily fluids as well, where accumulation of NETs has been shown to play a role in disease pathogenesis. For example, NETs can be quantified in the synovial fluid of joints to study rheumatoid arthritis (Corsiero et al., 2016) or excretion of NETs in urine in certain inflammatory conditions such as urinary tract infections (Yu et al., 2019). Since the smear assay requires only 1 μL of sample, this assay can be applied to samples that can be obtained only in smaller quantities; for example, NETs have shown to be involved in many ocular diseases such as glaucoma, age-related macular degeneration, dry eye, and diabetic retinopathy for which only a very small volume of ocular fluid can be obtained (Martínez-Alberquilla et al., 2022). Another example is bronchiolar lavage, where NETs have been shown to be elevated in patients with pneumonia-related acute respiratory distress syndrome (Bendib et al., 2019), primary graft dysfunction after lung transplantation (Sayah et al., 2015), and COVID-19 (Yaqinuddin et al., 2020). Moreover, these techniques can also be applied to pediatric autoimmune and inflammatory diseases such as pediatric lupus, where there are limitations on sample volume (Garcia-Romo et al., 2011).