Synovial fluid cell isolation protocol affects immune cell recovery and composition across inflammatory states
5 affiliations
- 1Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
- 2Department of Biology, Chair of Genetics, Friedrich-Alexander-Universitat Erlangen-Nurnberg (FAU), Erlangen, Germany
- 3The REMEDY Center, Division of Rheumatology and Research, Diakonhjemmet Hospital, Oslo, Norway
- 4Department of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway
- 5Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
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.
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BibTeX
@article{tesfaye2026synovial,
title = {{Synovial fluid cell isolation protocol affects immune cell recovery and composition across inflammatory states}},
author = {Abel Mekonnen Tesfaye and Jara Tsegaye Leta and Danh Phung and Erlend Holm-Nordli and Julie Røkke Osen and Josefine Eilsø Nielsen and Hilde Berner Hammer and Andreas Lossius and Guro Løvik Goll and Silje Watterdal Syversen and Asbjørn Christophersen},
journal = {RMD Open},
year = {2026},
month = oct,
doi = {10.1136/rmdopen-2026-007291},
url = {https://doi.org/10.1136/rmdopen-2026-007291},
note = {CC BY-NC 4.0},
}
RIS
TY - JOUR
TI - Synovial fluid cell isolation protocol affects immune cell recovery and composition across inflammatory states
AU - Abel Mekonnen Tesfaye
AU - Jara Tsegaye Leta
AU - Danh Phung
AU - Erlend Holm-Nordli
AU - Julie Røkke Osen
AU - Josefine Eilsø Nielsen
AU - Hilde Berner Hammer
AU - Andreas Lossius
AU - Guro Løvik Goll
AU - Silje Watterdal Syversen
AU - Asbjørn Christophersen
PY - 2026
DA - 2026/10/23
JO - RMD Open
DO - 10.1136/rmdopen-2026-007291
UR - https://doi.org/10.1136/rmdopen-2026-007291
AB - 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.
SN - 2056-5933
ER -