Methods to Assess Proliferation of Stimulated Human Lymphocytes In Vitro: A Narrative Review

Abstract

The ability to monitor lymphocyte responses is critical for developing our understanding of the immune response in humans. In the current clinical setting, relying on the metabolic incorporation of [3H] thymidine into cellular DNA via a lymphocyte proliferation test (LPT) is the only method that is routinely performed to determine cell proliferation. However, techniques that measure DNA synthesis with a radioactive material such as [3H] thymidine are intrinsically more sensitive to the different stages of the cell cycle, which could lead to over-analyses and the subsequent inaccurate interpretation of the information provided. With cell proliferation assays, the output should preferably provide a direct and accurate measurement of the number of actively dividing cells, regardless of the stimuli properties or length of exposure. In fact, an ideal technique should have the capacity to measure lymphocyte responses on both a quantitative level, i.e., cumulative magnitude of lymphoproliferative response, and a qualitative level, i.e., phenotypical and functional characterization of stimulated immune cells. There are many LPT alternatives currently available to measure various aspects of cell proliferation. Of the nine techniques discussed, we noted that the majority of these LPT alternatives measure lymphocyte proliferation using flow cytometry. Across some of these alternatives, the covalent labelling of cells with a high fluorescence intensity and low variance with minimal cell toxicity while maximizing the number of detectable cell divisions or magnitude of proliferation was achieved. Herein, we review the performance of these different LPT alternatives and address their compatibility with the [3H] thymidine LPT so as to identify the “best” alternative to the [3H] thymidine LPT.

Effects of A1 Milk, A2 Milk and the Opioid-like Peptide β-Casomorphin-7 on the Proliferation of Human Peripheral Blood Mononuclear Cells

“…Radioactivity of each well was quantified using a β-scintillation counter MicroBeta 2 (PerkinElmer), presenting measurements in counts per minute (cpm). The degree of cellular uptake of [ 3 H]-thymidine is indicative of cells undergoing DNA replication in the S-phase of the cell cycle and therefore represents cell proliferation [ 31 ].…”

Developmental Exposure To Low Doses Of Dichlorodiphenyltrichloroethane Disrupts Functional And Morphologic Maturation Of The Spleen In Prepubertal Rats

“…It is known that primed lymphocytes transform into blasts and proliferate after repeated administration of antigen. The in vitro transformation reaction of blasts in response to mitogens and the assessment of the proliferative response allows assessing the functional potential of lymphocytes [14,15]. In the present study, we focused on assessing the proliferative response of splenic lymphocytes to the T cell mitogen concanavalin A and evaluating splenic morphology in prepubertal rats exposed to DDT during prenatal and postnatal development.…”