SAT0028 The importance of proper handling of human synovial fluid for arthritis research
Authors: H. Brouwers, J.H. von Hegedus, R.E.M. Toes, T.W.J. Huizinga, M.A. Giera, M. Kloppenburg, A. Ioan-Facsinay
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Synovial fluid (SF) is commonly used for diagnostic and research purposes since it is easily obtained and is thought to represent the local inflammatory environment. Common practice to obtain insight in the disease pathogenesis of arthritic diseases is the characterisation and quantification of synovial fluid cells and the measurement of inflammatory mediators. SF has a complex composition, containing hyaluronic acid which makes the fluid viscous and non-homogeneous, hampering subsequent analyses. Hyaluronic acid can be broken down by the addition of hyaluronidase, however, this is not commonly used in arthritic research. <h3>Objectives</h3> To determine the effect of hyaluronidase treatment on quantification and identification of SF cells and soluble mediators. <h3>Methods</h3> SF was obtained from twelve arthritis patients after knee aspirations as part of standard clinical care. Nine patients were diagnosed with rheumatoid arthritis, two with osteoarthritis and one with juvenile idiopathic arthritis. For cell analysis, synovial fluid was first centrifuged and the pellet was separated from the fluid. The fluid was subsequently treated with hyaluronidase and centrifuged again to isolate remaining cells. Cell numbers and phenotype were determined using flow cytometry. For soluble mediator measurements, 6–10 aliquots were taken and treated as represented in figure 1 resulting in set 1 and set 2. Set 1 contains replicates taken form SF before hyaluronidase treatment while set 2 mimics replicates which are taken after hyaluronidase treatment. Interleukin (IL)−8 was measured by ELISA and a total of seven fatty acid and oxidised fatty acid levels were determined using LC-MS/MS in all aliquots. <h3>Results</h3> Between 0.8%–70% of immune cells (median 5%) are lost when the SF is not treated with hyaluronidase. This percentage is higher for T and B cells: 7%–85% (median 22%) and 7%–88% (median 23%), respectively. To assess the variation between the soluble mediator concentrations in set 1 and set 2, the coefficients of variation (CV) of the replicate measurements were compared. Aliquots in set 2 showed a lower CV for the oxidised lipids 17-HDHA, leukotriene B<sub>4</sub> and prostaglandin E<sub>2</sub> for all patients tested. For IL-8, the oxidised lipid 15-HETE, and fatty acids arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), it did not matter whether the aliquots were taken before or after hyaluronidase treatment. While in general the average levels of the soluble mediators tested were comparable between set 1 and set 2, we did observe large differences in IL-8 and AA levels in two independent patients. <h3>Conclusions</h3> Up to 70% of the synovial fluid cells are missed in analysis when the SF is not treated with hyaluronidase before cell isolation, leading to erroneous conclusion especially when investigating rare cells populations like antigen specific B or T cells, when searching for novel cell populations, or when correlations are made between clinical parameters and cell numbers. In addition, some cytokine as well as lipid levels determined in SF without hyaluronidase treatment might not accurately reflect their actual concentrations in SF. <h3>Disclosure of Interest</h3> None declared