Introduction: Infections of the central nervous system (CNS) remain a major burden of disease. Cerebrospinal fluid (CSF) is an essential sample for the investigation of CNS pathologies and flow cytometry enables detailed immunophenotyping of cells present in CSF.However, the pauci-cellular nature of CSF, and the rapid cell death following sampling, incumbers the use of flow cytometric analysis of these samples. Immediate processing and analysis of CSF for flow cytometry is not feasible in busy clinical environments where sample collection is unpredictable, and flow cytometers are not readily available. Therefore, developing a method to easily store CSF samples is highly desirable for clinically relevant research on CNS pathologies. Thus, the objective of this study was to examine 2 methods of long-term storage of CSF samples which ensured reliable measurement of cell percentages and relative proportion of cell subsets using flow cytometry. Aims: To examine percentages and relative proportion of subsets of selected peripheral leukocytes and brain derived cells in 1) cryopreserved CSF in comparison to freshly processed CSF, and 2) Transfix-treated CSF in comparison to freshly processed CSF. Method: CSF samples were prospectively collected and processed as follows 1) Freshwithin 24 hours (the current gold standard); 2) Cryopreserved- analysed after 1 month storage at..............Percentages of numerous white blood cell populations and brain-derived immune cells were analysed using flow cytometry and compared across these methods. The median fluorescent intensity of select markers was also compared across these methods. Results: The majority of cell percentages were not statistically significantly different between Fresh and Cryopreserved CSF, and cell proportions were comparable. Conversely, loss of marker expression of various lymphocyte sub-populations was observed in Transfixtreated CSF compared to Fresh, and certain cell populations could not be clearly distinguished in Transfix-treated CSF. Conclusion: Cryopreservation is a feasible option for long-term storage of CSF and allows quantification of cell percentages and immunophenotyping of peripheral and brain-derived cell populations by flow cytometry. This offers valuable opportunities for clinical research across a broad spectrum of CNS conditions (infectious and non-infectious). Further, this work highlights the potential to cryopreserve other surgical specimens for which the application of flow cytometry is currently limited by resource constraints and low cell counts.
