Table 4 Summary of recommendations for sample collection, processing, and storage
Step | Sample type | Key recommendations | Other recommendations |
|---|---|---|---|
Participant Preparation | |||
Blood | Overnight fasting when appropriate. | ||
CSF | Adequate hydration. | ||
Faecal | Instruct to avoid urine contamination; record bowel symptoms and laxative use. | ||
Saliva | No food, drink, smoking, or brushing 1 h before; rinse mouth 10 min before; record oral health status. | ||
Collection method | |||
Blood | Collect in appropriate tubes (EDTA, heparin, citrate, plain). | Tube choice affects downstream analyses | |
PBMCs | Tubes containing anticoagulants are recommended: heparin standard, EDTA higher yield, Citrate (ACD or CPD) preserves functionality; collect 10–50 mL | Standard Ficoll isolation yields 0.5–2 million PBMCs/mL; adjust blood volume according to assay needs. | |
Serum | Collect blood in plain tubes. | ||
Plasma | Collect blood in EDTA tubes. | Plasma is preferred for low-abundance cytokines | |
CSF | Collect 5–10 mL; discard the first five drops; use atraumatic needle and polypropylene tubes. | Avoid blood contamination; exclude samples with >500 RBCs/μL. | |
Faecal | Collect 1–2 g in sterile containers | ||
Saliva | Use passive drool method; ≥200 µL. | ||
Timing of collection | |||
All | Prefer morning for blood collection; standardise timing when possible; always record collection time. | ||
Sample handling | |||
Blood | Define intended use before processing. | Ensures correct tube and protocol selection. | |
PBMCs | Isolate PBMCs using Ficoll-Paque, CPT, or Leucosep/Lymphoprep tubes; document anticoagulant type and isolation method; process within 8 h at room temperature; do not exceed 24 h before processing. | CPT/Leucosep simplify isolation and reduces variability; manual Ficoll with EDTA yields higher PBMC counts; delayed processing impairs viability; cell number requirements depend on downstream assays. | |
PMNCs | Use Percoll or sedimentation to isolate PMNCs; with limited blood sample availability, isolate PMNCs and PBMCs together via Ficoll-Leucosep, and combine post-isolation for immunophenotyping. | Percoll reduces neutrophil priming; neutrophils are highly sensitive to handling. | |
Serum | Allow to clot ≥15 min, centrifuge within 60 min at 1000–2000×g for 10–15 min at 4 °C. | Delays and tube material affect measurements; serum cytokine levels may rise due to platelet activation | |
Plasma | Centrifuge at 1000–2000×g for 10–15 min at 4 °C within 60 min of collection | ||
CSF | Keep on ice; centrifuge at 500–1500×g for 10–15 min 4 °C within 60 min. | Use the cell pellet immediately for immunophenotyping to maximise cell number | |
Faecal sample | Aliquot quickly | ||
Storage | Avoid multiple freeze-thaw cycles | ||
Blood | |||
PBMCs | Controlled-rate freezing with liquid nitrogen in 10% DMSO/FCS; concentrate PBMCs >6 × 10⁶ cells/mL | Fresh PBMCs preserve maximal functionality; cryopreservation enables batch analysis for longitudinal/multicenter studies; storage at –80 °C is detrimental to cell viability and genomic stability. | |
Serum | Use low-binding polypropylene tubes, store at –80 °C; use small aliquots to avoid freeze-thaw. | ||
Plasma | |||
CSF | Freeze pellet and supernatant at –80 °C. (*specific conditions are required for single-cell) | Pellet for RNA sequencing, for protein, extracellular vesicle, or cell-free RNA analysis | |
Faecal sample | Aliquot and freeze at –80 °C within 2 h; temporary 4 °C overnight allowed. Avoid freeze-thaw cycles. | ||
Saliva | Store at –80 °C in low-binding tubes in small aliquots (~200 µL). | ||
Documentation | All | Record collection time, processing time, deviations, and pre-analytical variables (e.g. medication, oral health). | Critical for reproducibility and adjustment of analyses |
Transport | All | Maintain cold chain using dry ice or equivalent. | Prevents sample degradation during transfer. |
