Table 1. Summary of practical considerations regarding sampling in TBM.

	Factors affecting quality	Blood	CSF
Proteins	Sensitive to freeze-thawing. Prepare aliquot plan to limit the number of cycles. Haemolysed samples may be problematic.	Collect in EDTA or heparin tubes. Centrifuge within 1 h at standardised speed (i.e. 1500–3000 × g) to obtain plasma. Store in polypropylene tubes at -80°C. Aim for ≥ 100μl aliquots.	Collect preferably in polypropylene tubes. Centrifuge within 1 h at standardised speed (i.e. 1500–3000 x g) to obtain supernatant while pelleting remaining erythrocytes and leukocytes. Store in polypropylene tubes at -80°C. Aim for ≥100μl aliquots.
Metabolites	After collection, store cool (4°C) till first centrifugation performed within 1 h. Avoid samples that appear haemolysed after centrifugation.	Collect in EDTA or heparin tubes. Centrifuge within 1 h at standardised speed (1500–3000 × g) to obtain plasma. Serum is second best. Store in polypropylene tubes at -80°C. Aim for ≥ 200μl aliquots.	Collect preferably in polypropylene tubes. Centrifuge within 1 h at standardised speed (1500–3000 × g) to obtain supernatant while pelleting remaining erythrocytes and leukocytes. Store in polypropylene tubes at -80°C. Discard samples with erythrocytes >500/μl. Aim for ≥200μl aliquots.
Pharmacokinetics	Short specimen transfer using cool box and protected from light. Maximum time to processing depends on drug, isoniazid is especially unstable.	Collect in heparin or EDTA tubes for plasma or clotted for serum assays. Centrifuge at standardised speed (1500–3000 × g for 10–15 minutes). Store in cryotubes at -80°C. Aim for ≥200μl aliquots.	Collect in cryovials directly or transfer to cryovials immediately after collection. Centrifugation not essential Store in cryotubes at -80°C. Aim for ≥200μl aliquots.
Flow cytometry	If possible, perform on fresh cells as fixation can influence expression of activation markers.	Collect in heparin or EDTA-tubes. No centrifugation until processing Store at room temperature until processing <24 h. Aim for ≥100μl blood per panel.	Collect in standard tubes Centrifuge gently (i.e. 300 × g) immediately (< 1 h) after collection Resuspend pellet in RPMI supplemented with BSA. Store at 4°C until processing <24 h. Aim for ≥500μl CSF per panel.
Transcriptomics	Early stabilisation is paramount	Collect in PAXgene tubes according to manufacturer's instructions. Aim to fill tube to maximum (2.5 mL) volume. No centrifugation. Shake tube, leave at room temperature for ≥2 h. Store at -80°C up to two years.	Collect in PAXgene tubes and process as with blood. Alternatively, collect in polypropylene tubes, centrifuge gently (800 × g for 10 minutes), add 500μl Tri-Reagent or TRIzol to pellet, mix vigorously and leave for 10 minutes at room temperature before freezing. Store blood PAXgene tubes at -80°C up to two years. No data exists on processing CSF RNA samples after more than six months.
Ex vivo cytokine production		Collect in heparin tubes for whole blood stimulation assays or in EDTA-tubes for subsequent PBMC isolation. No centrifugation. Store at room temperature till processing in <24 h.	N/A
Microbiological studies	Perform within 24 h after lumbar puncture.	N/A	Collect >6ml in sterile tubes. Centrifuge 10–15 minutes at 3000 × g.

TBM, tuberculous meningitis; CSF, cerebrospinal fluid; h, hour; EDTA, ethylenediaminetetraacetic acid; PBMC, peripheral blood mononuclear cells; RPMI, Roswell Park Memorial Institute medium; BSA, bovine serum albumin.