Research paperAn optimized protocol for adenosine triphosphate quantification in T lymphocytes of lymphopenic patients

•ATP concentration in T lymphocytes is proposed to reflect immune competence.•We optimized the intra-lymphocyte ATP dosage protocol for possible use in lymphopenic patients.•50,000 T cells are sufficient for intra-cellular ATP measurement.•Incubation duration of the assay can be drastically shortened, up to 30 min.•This functional testing deserves to be assessed in clinical studies.

In several clinical contexts, the measurement of ATP concentration in T lymphocytes has been proposed as a biomarker of immune status, predictive of secondary infections. However, the use of such biomarker in lymphopenic patients requires some adaptations in the ATP dosage protocol. We used blood from healthy volunteers to determine the optimal experimental settings. We investigated technical aspects such as the type of anticoagulant for blood sampling, the effect of freeze and thaw cycles, the reagent and sample mixing sequence, and the optimal dilution buffer. We also shortened the incubation time to 8 h, and even showed that a 30 min incubation may be sufficient. To evaluate the ATP rise upon lymphocyte activation, the optimal dose of stimulant was defined to be 4 μg/mL of phytohaemagglutinin. Lastly, we determined that the number of T cells needed for this measurement was as low as 50,000, which is compatible with the existing lymphopenia in clinical settings. This optimized protocol appears ready to be assessed in lymphopenic patients to further investigate the interconnection between T lymphocyte metabolism and impaired phenotype and functions.