Work

Synovial fluid cell isolation protocol affects immune cell recovery and composition across inflammatory states

Year: 2026

Type: article

Abstract: Objective Synovial fluid single-cell immunoprofiling is a powerful tool for investigating joint arthropathies. However, cell isolation protocols are yet to be standardised. We compared different cell-isolation protocols across diverse synovial fluid samples, evaluating cell recovery, sample inclusion rate and immune-cell composition.Methods From 48 synovial fluid samples with volumes>2 mL, we performed paired cell isolation of whole synovial fluid cells (WSFCs), with and without hyaluronidase and synovial fluid mononuclear cells (SFMCs) by density-gradient centrifugation. Of these, nineteen paired samples underwent deep immunophenotyping using a 31-marker spectral flow cytometry panel with dimensionality reduction and unsupervised clustering. Immune cell frequencies were quantified across protocols. Synovial fluid rheology was assessed in an additional 23 samples.Results WSFC isolation yielded higher cell recovery and lower sample loss than SFMC isolation, with differences varying with inflammation level. SFMC isolation reduced the sample inclusion rate (the proportion of samples with sufficient cell numbers for downstream profiling) by 50% in low-grade and 10% in high-grade inflammatory samples. Cell recovery correlated with synovial fluid viscosity. Repeated centrifugation alone matched the cell recovery seen with hyaluronidase treatment. Deep immunophenotyping revealed systematic protocol-dependent differences: WSFCs were enriched for CD8 +, CD4+ and γδ T cells, while SFMCs contained higher proportions of dendritic cells and classical monocytes. Both methods stratified samples by inflammation, but WSFCs provided superior cellular resolution.Conclusions SFMC isolation excluded low-grade inflammatory samples, introducing bias and reducing statistical power. WSFC-based approaches with hyaluronidase or repeated centrifugation improved recovery and are therefore recommended for synovial fluid immunoprofiling studies.

Source: RMD Open

DOI: 10.1136/rmdopen-2026-007291