Rapid one-step protocol for exploring immune cells in cerebrospinal fluid.
Author(s): Wallisky Millet E, Malergue F, Petrirena Hernandez G, Boucraut J
Publication: J Neuroimmunol, 2025, Vol. 407, Page 578687
PubMed ID: 40684654 PubMed Review Paper? No
Purpose of Paper
This paper compared immune cell populations in stained blood and cerebrospinal fluid (CSF) specimens that were analyzed by flow cytometry immediately and after storage for up to 3 days at room temperature. Effects of storing CSF for up to 4 h at room temperature prior to staining with a one-step protocol on immune cell populations and apoptotic markers were also investigated.
Conclusion of Paper
There was no effect of storing stained blood specimens at room temperature for 3 days on the mean fluorescence intensity (MFI) or counts of CD45+ cells, T-cells, NK cells and monocytes, but leukocyte (P=0.01555) and T lymphocyte (P=0.004950) counts were slightly lower after 3 days. In CSF specimens, neither cell counts nor MFI were affected by post-staining storage. Overall, T-cell counts were not affected by pre-staining storage of CSF at room temperature for up to 4 h. However, in CSF from three of the four volunteers, monocyte counts decreased by 20% and 50% after pre-staining storage for 2 and 3 h, respectively. Interestingly, monocyte counts only decreased in the final CSF specimen when stored pre-staining for 4 h and that CSF specimen was the only one with just CD14+ CD16- monocytes (no CD14+ CD16+ monocytes) at the initial timepoint (<1 h). As a result of the stable T-cell population and decreasing monocyte population in some specimens, the ratio of T-Cells (CD4+) to monocytes (CD14+) increased when stored prestaining for 2 h in CSF from three of the four patients. Only one of the four CSF specimens had positively stained T-cells for apoptotic markers, and that was limited to Annexin V (AV, early apoptosis) staining in 5% of T-cells. In contrast, three of the four CSF specimens at 0 h had AV staining without propidium Iodide (AV+PI-) staining in >20% of monocytes, indicating early apoptosis, and AV+PI+ staining, indicating late apoptosis in <10% of monocytes. However, after the first timepoint, the percentage of AV+PI- and AV+PI+ increased in CSF of only one patient. The authors show that additional monocytes from CSF were lost due to adhesion to the tube wall.
Studies
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Study Purpose
This study compared immune cell populations in stained blood and cerebrospinal fluid (CSF) specimens that were analyzed by flow cytometry immediately and after storage for up to 3 days at room temperature. The effects of storing CSF for up to 4 h at room temperature prior to staining with a one-step protocol on immune cell populations and apoptotic markers were also investigated. EDTA blood was collected from four healthy volunteers. Immune cells were labeled with antibodies using the IM phenotyping BASIC dry panel, fixed with formaldehyde and stored at room temperature. Cell counts were analyzed by flow cytometry 0, 1, 2 or 3 days after staining. CSF was obtained within 1 h of lumbar puncture from 5 patients (diagnosis not specified) and, unless otherwise specified, was stained and fixed like the blood specimens but with the addition of antibodies against a red blood cell marker (CD235a) and an isotype control (IgG1). Cell counts were analyzed in both specimen types by flow cytometry 0, 1, 2 or 3 days after staining. To investigate the effects of a processing delay, aliquots of CSF from 4 patients were stored for <1 h or for an additional 1, 2, 3 and 4 h before staining. CSF aliquots stored for <1 h, 2 and 4 h were stained for annexin V and with propidium iodide to investigate if changes in cell populations could be attributed to apoptosis. To investigate potential cell loss due to adhesion to the tube wall, trypsin EDTA solution and DMEM were added to the tube after removal of CSF to release cells, which were then analyzed in the same way as the CSF specimens.
Summary of Findings:
There was no effect of storing stained blood specimens for 3 days at room temperature on the mean fluorescence intensity (MFI) or counts of CD45+ cells, T-cells, NK cells and monocytes, but leukocyte (P=0.01555) and T lymphocyte (P=0.004950) counts were slightly lower after 3 days. In CSF specimens, neither cell counts nor MFI were affected by storage after staining. Overall, T-cell counts were not affected by pre-staining storage of CSF. However, in CSF from three of the four volunteers, monocyte counts decreased by 20% and 50% after pre-staining storage for an additional 2 and 3 h, respectively. Interestingly, monocyte counts only decreased when the final CSF specimen was stored for an additional 4 h and that CSF specimen was the only one with just CD14+ CD16- monocytes (no CD14+ CD16+ monocytes) at the initial timepoint (<1 h). As a result of the stable T-cell population and decreasing monocyte population in some specimens, the ratio of T-Cells (CD4+) to monocytes (CD14+) increased, starting at 2 h in CSF from three of the four patients. Only one of the four CSF specimens had any positively stained T-cells for apoptotic markers, which was limited to Annexin V (AV, early apoptosis) staining in 5% of T-cells. In contrast, three of the four CSF specimens at 0 h had AV staining without propidium Iodide (AV+PI-) staining in >20% of monocytes, indicating early apoptosis, and AV+PI+ staining, indicating late apoptosis in <10% of monocytes. However, after the first timepoint, the percentage of AV+PI- and AV+PI+ increased in the CSF specimen of only one patient. The authors show that additional monocytes from CSF were lost due to adhesion to the tube wall after only 3 h of prestaining storage (earliest timepoint).
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 h (<1 h)
+1 h
+2 h
+3 h
+4 h
Storage Time at room temperature 0 days
1 day
2 days
3 days