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.

The process of granuloma formation is complex, and due to species differences, the validity of animal studies is somewhat questioned. Moreover, the large number of animals needed to observe the different stages of development also raises ethical questions. Therefore, researchers have explored the use of human peripheral blood mononuclear cells (PBMCs), a heterogeneous population of immune cells, in an in vitro model. This review included in vitro studies that focused on exposing PBMCs—from healthy, sensitized, or diseased individuals—to antigens derived from infectious agents—such as mycobacteria or Schistosoma spp.—or inorganic antigens—such as beryllium. The reviewed studies mainly explored how human in vitro granuloma models can contribute towards understanding the pathogenesis of granulomatous diseases, especially during the early stages of granuloma formation. The feasibility of granuloma modelling was thus largely assessed via experimental techniques including (1) granuloma scoring indices (GI), (2) cell surface markers and (3) cytokine secretion profiling. While granuloma scoring showed some similarities between studies, a large variability of culture conditions and endpoints measured have been identified. The lack of any standardization currently impedes the success of a human in vitro granuloma model.

The detection of antigen specific lymphocyte responses plays a vital role in the diagnosis of various diseases. Beryllium-specific [³H] thymidine lymphocyte proliferation test (LPT) is regarded as a gold standard in identifying chronic beryllium disease (CBD) cases. Alternatively, flow cytometric based carboxyfluorescein succinimidyl ester (CFSE) assay, has several benefits as opposed to LPT, since it further permits both phenotypical characterization and functional analysis of proliferating lymphocyte subsets.

Introduction: Silicosis is caused by (occupational) exposure to silica particles. There is a general lack of knowledge on the immunologic pathways in the pathogenesis of early-stage silicosis. By stimulating peripheral blood mononuclear cells (PBMCs) with silica particles, we aimed to identify the cytokines secretion profile of silicosis ex vivo. Concurrently, we also measured the proliferative responses of PBMCs upon silica stimulation. Methods: PBMCs from silicotic and healthy subjects were stimulated with both colloidal silica (Ludox) and synthetic amorphous silica (NM-200) at a concentration of 10 µg/ml and four 10-fold serial dilutions. Following 7 days of exposure, supernatant and cells were collected for multiplex assay and lymphocyte proliferation analysis, respectively. Proliferation was assessed by stimulation index (SI; considered positive when >2.5). Results: Without a stimulus, cytokine levels did not differ significantly between both groups. Across all concentrations of both silicas, increased release of pro-inflammatory cytokines IL-1β, IFN-γ and reduced release of TNF-α were noted in silicotic subjects when compared with healthy subjects. Within the silicotic patients, Ludox and NM-200 induced a different pattern of IL-12p70, IL-17a,IL-10 and IL-4 release. Across higher concentrations of 1 and 10 µg/ml, mean SI values for T (CD3⁺) and B (CD19⁺) cells combined were positive among silicotic patients, unlike healthy subjects. Considering only B cells, positive SI values were found in silicotic patients across all concentrations. Conclusion: Both cytokine analysis and lymphocyte proliferation results allowed for silicotic subjects to be distinguished from healthy subjects.