Background/Purpose: Recent data show that neutrophils are able to release chromatin and granular enzymes in web-like structures called neutrophil extracellular traps (NETs). The release of NETs has a physiological role in the innate immunity response by trapping microbes. However, further data indicate that neutrophils are able to release NETs under different conditions such as in the presence of monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals. In particular, it has been postulated that an increasing release of NETs up to a critic threshold during gout attack may lead to the rapid resolution of the inflammatory phase of the disease by packaging MSU crystals and degrading proinflammatory cytokines. Nowadays, the gold standard for the diagnosis of crystal-induced arthropathies remains the identification of crystals in synovial fluid. Interestingly, at standard wet synovial fluid analysis, besides cells and crystals, clumps of non-refractile fibril-like material are commonly observed. These fibrillar aggregates, primarily composed by fibrin and collagen fibrils, appear as ragged and angulated meshes where crystals remain often trapped, making their identification easier. Since crystals appear concentrated on these networks, we found intriguing to understand whether these clumps of non-refractile fibril-like material may contain NETs.

Methods: Synovial fluid samples from MSU and CPPD arthropathies (n=3 for each group) were collected for wet analysis by polarizing, compensated microscopy and then immediately fixed and processed for fluorescence microscopy. Samples were stained on poly-L-lysine-coated glass coverslips with DAPI nucleic acid stain and with anti-neutrophil elastase (NE, rabbit polyclonal) and anti-histone H3 citrullinated antibodies (H3, mouse monoclonal) (Abcam, UK and LS-Bio, US, respectively) in order to visualize NETs’ components.

Results:  The definitive diagnosis of crystal-induced arthropathy was confirmed by the identification of MSU or CPPD crystals in synovial fluid. In addition, fibril-like aggregates trapping crystals and cells were observed in all the samples analyzed. After immunostaining, web-like NET structures containing DNA, NE and H3-histone were visualized by immunofluorescence. NET structures appeared as networks trapping MSU or CPPD crystals.

Conclusion:  NETs can be visualized in synovial fluid from MSU and CCPD crystal-induced arthropathies. NETs appear as part of the clots of fibril-like material frequently observed in synovial fluid trapping crystals