Stabilization of leukocytes from cerebrospinal fluid for central immunophenotypic evaluation in multicenter clinical trials

Abstract

Analysis of cerebrospinal fluid (CSF) represents a valuable window into the pathogenesis of neuroinflammatory diseases, such as multiple sclerosis (MS). However, analysis of the cellular fraction of CSF is often neglected because CSF cells die rapidly ex vivo. Immunophenotyping of CSF cells in multicenter clinical trials requires sample preservation and shipping to a centralized lab. Yet, there is no consensus on the best method to preserve intact CSF cells and no detailed evaluation of subset-specific cell loss.We used flow cytometry to compare major leukocyte populations in fresh CSF (processed within 2 h) to cells fixed for 48 h with TransFix-EDTA® or cryopreserved and thawed after 96 h. We observed a statistically significant loss of total mononuclear cells, total T cells, CD3+ CD8- T cells, and CD3+ CD8+ T cells after cryopreservation compared to fresh or fixed (p < 0.001), with no significant difference between fresh and fixed. Thus, our results demonstrate that TransFix-EDTA® was superior to cryopreservation for preserving intact CSF T cells. Surprisingly, neither cryopreservation nor fixation had a significant effect on recovery of low frequency cell subsets in CSF, including B cells, NK cells, NKT-like cells, CD14+ monocytes, or CD123+ DCs, versus fresh CSF.To determine the effect of prolonged fixation on cell recovery, we analyzed major CSF cell subsets by flow cytometry after 24, 48, or 72 h of fixation with TransFix-EDTA®. We observed a consistent and progressive loss in the absolute counts of all subsets over time, although this effect was not statistically significant.We conclude that for immunophenotyping of major CSF cell subsets by flow cytometry, fixation with TransFix-EDTA®, shipment to a central lab, and analysis within 48 h is a feasible method to ensure stability of both absolute cell number and relative frequency. This method is a valuable alternative to fresh CSF analysis and can be implemented in multicenter clinical trials.