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The influence of fixation and cryopreservation of cerebrospinal fluid on antigen expression and cell percentages by flow cytometric analysis.

Author(s): Singh G, van Laarhoven A, Adams R, Reid TD, Combrinck J, van Dorp S, Riou C, Thango N, Enslin J, Kruger S, Figaji AA, Rohlwink UK

Publication: Sci Rep, 2024, Vol. 14, Page 2463

PubMed ID: 38291295 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the relative percentages of immune cells and MFI in fresh, cryopreserved, and Transfix CSF specimens collected from pediatric patients diagnosed with a CNS infection and in PBMC-spiked pediatric CSF. Cell type percentages and MFI were also compared among fresh CSF and CSF that was stored at 2-8°Cin Transfix for 24 h, 1 week, or 2 weeks. The initial study included a total of seven ventricular CSF specimens and two lumbar CSF specimens that were collected during neurosurgery from five pediatric patients (3 males and 2 females; 3 diagnosed with tuberculous meningitis and three diagnosed with bacterial meningitis). The second cohort consisted of 20 ventricular CSF specimens that were collected from 13 patients undergoing neurosurgery without CNS infections. Specimens from the second cohort were spiked with cryopreserved PBMCs that were previously isolated from healthy adults to achieve one million cells per sample. All CSF specimens were divided into three aliquots which were: 1) immediately mixed with RPMI media containing 10% fetal bovine serum and placed at 4°C for < 1 h (Fresh);  2) immediately transferred to a Transfix tube and stored at 2-8°C for 24 h, 1 week, or 2 weeks (Transfix CSF); and 3) immediately centrifuged at 300 g for 5 min at 4°C, resuspended in RPMI, supplemented with cryo-solution (FBS and DMSO), transferred to a cryotube in a Mr Frosty freezing container and stored at -80°C for 1 month (Cryopreserved CSF).  Following incubations, cells were pelleted at 300 g for 5 min at 4°C, and the cell pellet was resuspensed in a 16-color antibody cocktail and processed for flow cytometry.

   Summary of Findings:

   In non-spiked specimens, the percentages of each cell population and percentage of cells expressing activation markers were comparable in fresh CSF and cryopreserved CSF. In contrast, the cryopreserved spiked CSF had fewer viable cells (43.3% versus 47.15%, P=0.05), CD45+CD11b+ cells (86.25% versus 91.25%, P=0.01), and CD3+ cells (69.3% versus 72.75%, P=0.02) and more CD11b++ cells (14.35% versus 9.34%, P=0.01) than fresh specimens. Transfix CSF from the non-spiked specimens that were stored for 2 weeks had fewer CD11b++ cells (0.028% versus 2.64% P=0.01), CD4+ cells (47.7% versus 69.1%, P=0.001), CD8+ cells (12% versus 19.6%, P=0.007), NK cells (18% versus 62.3%, P=0.004), and non-classical monocytes (0.07% versus 34.2% P=0.03) and fewer cells expressing the CD69+ activation marker (3.25% versus 41.2%, P=0.001) than fresh cells. In the spiked specimens, Transfix CSF stored for 2 weeks also had fewer CD11b++ cells (0.021% versus 9.34%, P<0.001), CD4+ cells (47.6% versus 87.1%, P<0.001), NK cells (3.64% versus 32.5%, P<0.001), and non-classical monocytes (0.69% versus 58.6%, P<0.001) and fewer cells expressing the CD69+ activation marker (0.02%% versus 0.98%, P<0.001) than fresh specimens, but also had fewer CD3+cells (60.7% versus 72.75%, P<0.001), CD161+ cells (4.27% versus 10.9%, P<0.01), MAIT cells (1.39% versus 6.38%, P=0.007), and HLA-DR+ cells (0.91% versus 2.21%, P<0.001) and more γδ TCR+ cells (1.95% versus 0.48%, P=0.01), and classical monocytes (7.75% versus 1.12%, P<0.001) than fresh cells.  Importantly, populations of CD45+CD11b+ cells, Va7,2+ cells, and B-cells were not distinguishable from one another in Transfix CSF that was stored for 1 or 2 weeks and thus could not be analyzed, but were identifiable in Transfix CSF that was stored for 24 h. CSF stored in Transfix for 24 h had higher percentages of CD45+cells (P=0.05), CD11b++cell (P=0.046), CD161+ cells (P=0.046), and MAIT cells (P=0.05), all of which were lower in Transfix CSF specimens stored for 2 weeks relative to fresh specimens. The agreement between fresh and cryopreserved CSF was higher than the agreement between fresh and transfix CSF in both non-spiked specimens (mean of differences 3.19 and 14.82, respectively) and spiked specimens (mean of differences 0.13 and 12.38, respectively). Cell percentages were very strongly correlated between fresh and cryopreserved CSF (r=0.91, P<0.001 for non-spiked CSF and r=0.97, P<0.001 for spiked CSF) and strongly correlated between fresh and Transfix CSF (r=0.81, P<0.001 for both non-spiked CSF and spiked CSF). Notably, the mean fluorescent intensity (MFI) of CD19+ was lower in both cryopreserved and Transfix CSF than fresh CSF regardless of spiking (P<0.05 all); CD45 MFI was lower in spiked cryopreserved and Transfix CSF than fresh CSF (P=0.001 and P=0.009), CD3 MFI was higher in Transfix CSF than fresh CSF in spiked and non-spiked specimens (P=0.001, both), and CD11b MFI was higher in spiked cryopreserved and Transfix CSF than fresh CSF (P=0.021, and P<0.001).

   Biospecimens

   • Bodily Fluid - Cerebrospinal Fluid

   Preservative Types

   • Frozen
   • None (Fresh)
   • Other Preservative

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   Protein	Flow cytometry
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen Transfix
   Preaquisition	Diagnosis/ patient condition	CNS infections No CNS infections (PBMC-spiked)
   Biospecimen Preservation	Time in fixative	24 h 1 week 2 weeks

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