Center for Neuroinflammation and Experimental Therapeutics and the Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Immune Tolerance Network, Bethesda, MD, USA.
J Immunol Methods. 2022 Nov;510:113344. doi: 10.1016/j.jim.2022.113344. Epub 2022 Aug 28.
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.
分析脑脊液（CSF）是了解神经炎症性疾病（如多发性硬化症[MS]）发病机制的重要窗口。然而，由于 CSF 细胞在体外迅速死亡，对 CSF 细胞的细胞成分进行分析往往被忽视。在多中心临床试验中对 CSF 细胞进行免疫表型分析需要保存样本并运送到集中的实验室。然而，目前尚无关于保存完整 CSF 细胞的最佳方法的共识，也没有对特定细胞亚群丢失的详细评估。我们使用流式细胞术比较了新鲜 CSF（在 2 小时内处理）中的主要白细胞群与用 TransFix-EDTA®固定 48 小时或冷冻保存并在 96 小时后解冻的细胞。我们观察到与新鲜或固定相比，冷冻保存后总单核细胞、总 T 细胞、CD3+CD8-T 细胞和 CD3+CD8+T 细胞的数量均有统计学显著下降（p<0.001），而新鲜和固定之间无显著差异。因此，我们的结果表明，与冷冻保存相比，TransFix-EDTA®更适合用于保存完整的 CSF T 细胞。令人惊讶的是，冷冻保存或固定对 CSF 中低频细胞亚群的恢复均无显著影响，包括 B 细胞、NK 细胞、NKT 样细胞、CD14+单核细胞或 CD123+DC 细胞，与新鲜 CSF 相比。为了确定长时间固定对细胞恢复的影响，我们用 TransFix-EDTA®固定 24、48 或 72 小时后，通过流式细胞术分析主要 CSF 细胞亚群。我们观察到随着时间的推移，所有亚群的绝对计数都持续且逐渐减少，尽管这种影响没有统计学意义。我们得出的结论是，对于通过流式细胞术对主要 CSF 细胞亚群进行免疫表型分析，使用 TransFix-EDTA®固定、运送到中央实验室并在 48 小时内分析是一种可行的方法，可确保绝对细胞数和相对频率的稳定性。该方法是新鲜 CSF 分析的有效替代方法，可在多中心临床试验中实施。
