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Methods to Assess Proliferation of Stimulated Human Lymphocytes In Vitro: A Narrative Review
Nirosha Ganesan1,2, Steven Ronsmans1,3, Peter Hoet1,2
1Laboratory of Toxicology, Unit of Environment & Health, Department of Public Health and Primary Care, KU Leuven, 3000 Leuven, Belgium.
Insights
The [3H] thymidine lymphocyte proliferation test (LPT) has limitations. This review evaluates alternative methods, many using flow cytometry, to accurately measure lymphocyte proliferation and immune responses.
Area of Science:
- Immunology
- Cell Biology
Background:
- Monitoring lymphocyte responses is crucial for understanding human immunity.
- The current standard, [3H] thymidine lymphocyte proliferation test (LPT), measures DNA synthesis but can be affected by cell cycle stages, leading to interpretation issues.
Purpose of the Study:
- To review and compare alternative methods for measuring lymphocyte proliferation.
- To identify the optimal alternative to the [3H] thymidine LPT for accurate immune response assessment.
Main Methods:
- A review of nine alternative lymphocyte proliferation assays.
- Focus on methods utilizing flow cytometry for cell proliferation measurement.
- Evaluation of techniques based on covalent cell labeling for high fluorescence intensity, low variance, minimal toxicity, and accurate proliferation measurement.
Main Results:
- Most reviewed LPT alternatives employ flow cytometry.
- Several alternatives achieve covalent cell labeling with high fluorescence and low variance, minimizing toxicity.
- These methods aim to maximize detectable cell divisions and proliferation magnitude.
Conclusions:
- Alternative methods, particularly flow cytometry-based assays, offer advantages over the traditional [3H] thymidine LPT.
- The ideal assay should provide quantitative and qualitative measurements of lymphocyte responses.
- Further evaluation is needed to determine the best compatible alternative to the [3H] thymidine LPT.
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.
