Background: Single-cell genome sequencing and proteomic analysis using high-parameter cytometry from synovial biopsy, blood, and synovial fluid are advancing precision rheumatology. Synovial fluid (SF) samples offer distinct advantages: they are more easily accessible than synovial biopsies and may represent tissue pathology more accurately than blood. However, viscoelastic properties and cell yields vary widely in SF samples, leading to diverse isolation protocols.

Objectives: This study evaluates cell yield, composition, and phenotype by comparing three widely used SF cell isolation protocols — simple centrifugation, density gradient centrifugation, and hyaluronidase treatment with centrifugation.

Methods: SF was collected from 73 arthritis patients at Diakonhjemmet Hospital. While inflammatory states exist on a spectrum, for practical purposes samples were classified as overtly inflamed (>2000 cells/mm³) or sub-clinically inflamed (<2000 cells/mm³). 32 samples with sufficient volume were processed using all three methods. Cell composition and phenotype were analyzed with a 37-color panel using spectral flow cytometry.

Results: In sub-clinically inflamed SF, density gradient centrifugation recovered 50% fewer cells compared to simple centrifugation or hyaluronidase treatment. Notably, the viscosity-lowering effect of hyaluronidase was significantly pronounced in sub-clinically inflamed SF where 5 - 30 % of cells were isolated after enzyme treatment. In contrast, overall cell yields were comparable across protocols in overtly inflamed SF, and hyaluronidase treatment offered little benefit. Preliminary spectral cytometry data revealed method-dependent differences in monocyte subsets (classical, non-classical, and intermediate), while maintaining consistent proportions of lymphocyte subsets (CD4+ T cells, CD8+ T cells, γδ T cells, NK cells, B cells) and disease-relevant phenotypes such as regulatory T cells and peripheral T helper cells.

Conclusion: The inflammatory state of SF influences fluid viscosity, cell yield, and monocyte composition. Density gradient centrifugation, though widely used, demonstrated poor cell recovery in sub-clinically inflamed samples. This leads to a selection bias where viable cells are predominantly derived from overtly inflamed SF. Consequently, sub-clinically inflamed states, which may provide critical insights into early disease mechanisms, could be systematically underrepresented in single-cell analysis studies. These findings highlight the importance of using isolation protocols tailored to the inflammatory state of SF to ensure comprehensive and representative single-cell analyses across the full spectrum of inflammation, from smoldering to overtly active disease.

REFERENCES: NIL.

Acknowledgements: We thank the patients who have donated biological material and The Flow Cytometry Core Facility at the Faculty of Medicine at the University of Oslo/Oslo University Hospital.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.