Table 3.

Basic protocol for in vitro determination of lymphocyte subpopulations and function.

(a) Determine the absolute count of the following lymphocyte subpopulations, and compare the results with age-matched reference values
CD3+	T lymphocytes
CD3+/CD4+	Helper-T lymphocytes
CD3+/CD8+	Cytotoxic T lymphocytes
CD3+/HLA-DR+	Activated T lymphocytes
CD3+/CD4–/CD8–	‘Double-negative’ T cells
CD3+/TCR-γδ+	Subset of T lymphocytes
CD19+ or CD20+	B lymphocytes
CD3–/CD16+ and/or CD56+	NK cells
(b) Determine the uptake of [3H]-thymidine (or CFSE or activation markers) and compare the results with − preferably − age-matched controls after stimulation with:		Mitogens (e.g. PHA, PMA + ionomycin, PWM)
Consider monoclonal antibodies (e.g. CD2 ± CD28, CD3 ± CD28)
Antigens (e.g. tetanus, after booster vaccination)
Consider allogeneic cells

(a) Can be performed in many hospitals; for correct interpretation of the results, the advice of an immunologist is highly recommended. (b) Collaboration with an immunologist and specialized laboratory is recommended.

Abbreviations: CD = cluster of differentiation, CFSE = carboxyfluorescein succinimidyl ester, HLA = human leucocyte antigen, NK = natural killer, PHA = phytohaemagglutinin, PMA = phorbol myristate acetate, PWM = pokeweed mitogen, TCR = T-cell receptor.