Table 2.

Recommended procedures in processing blood samples for T cell assays.

Step	Recommendations	Level of evidence	Major unknowns	T cell assays used for validation	References
Blood drawing	Fasting state	E		n.a.	[1–20,71,72]
	Same time of the day	E		Whole blood IFN-γ/IL-10 ELISA	[21,22]
	Vacuum tubes or syringes	D		IFN-γ ELISPOT, class I MMrs	[23]
	Sodium or lithium heparin	C		IFN-γ ELISPOT, IFN-γ ICS	[24–26]
	Minimum delay in processing (8–12 h)	C		IFN-γ ELISPOT	[23,24,27–30]
	Room tewmperature storage, gentle agitation	D		IFN-γ ELISPOT, class I MMrs	[23,25,29,32]
	Dilution if storage > 8 h	C		IFN-γ ELISPOT, class I MMrs	[23,29]
Blood shipping	Insulated containers	B		IFN-γ ELISPOT	[33,34]
PBMC preparation	Ficoll ± Leucosep® or CPT™ tubes	D		IFN-γ ELISPOT, class I MMrs	[23,36–38]
			Ficoll preparations?		[35]
	Washes with media containing human serum	D		IFN-γ ELISPOT, class I MMrs	[23]
			Whole blood versus PBMCs?		[26,40–42]
PBMC freezing			Freezing media?		[24,53,54]
			Medium temperature?		[35,47,56]
			Caspase inhibitors?		[55]
	PBMC concentration ≤ 3 × 107/ml	E		n.a.	[53]
	Cooling rate 1°C/min down to −70°C	E		n.a.
	Prompt transfer to liquid nitrogen (24–72 h)	E		n.a.	[24,53]
	Storage temperature ≤ −132°C (vapour or liquid nitrogen)	E		n.a.	[24,53]
PBMC shipping	Liquid nitrogen (vapour or liquid phase)	D		IFN-γ ELISPOT, IFN-γ ICS	[24]
Long-term preservation	Better if < 6 months	D		IFN-γ ICS	[57]
PBMC thawing	Rapid, 37°C thawing temperature	E		n.a.	[53]
	15–50 ml thawing volumes	E		n.a.	[53]
	280–450 g, 5–10 min centrifugation	E		n.a.	[53]
	DNase treatment if clumping	C		[3H]-TdR, cytokine ELISA	[58]
	1–12 h PBMC resting	C		IFN-γ ELISPOT	[27,46,59,60]
T cell assays	Human serum-supplemented media	E		n.a.
	Serum-free media	B		IFN-γ ELISPOT	[54]
			Addition of low-dose cytokines (e.g. IL-7)?		[33,34,54]
	High quality protein or peptide antigens	A		[3H]-TdR	[62–67]
	Positive controls: recall antigens and polyclonal stimuli	E		n.a.

[³H]-TdR: thymidine incorporation; ICS: intracellular cytokine staining; MMrs: human leucocyte antigen multimers; n.a.: not available; PBMC: peripheral blood mononuclear cells; IL: interleukin; IFN: interferon; ELISPOT: enzyme-linked immunospot; ELISA: enzyme-linked immunosorbent assay.