This is a preliminary study, where we included only 8 study subjects to know whether we can use 1:5 and 1:10 diluted whole blood assays or not. Healthy laboratory volunteers were enrolled in this study because their exposure to M. tuberculosis, would have made them possess sensitized T cells, which upon re-stimulation with M. tuberculosis specific antigens, will induce immune response and secrete cytokines. Since M. tuberculosis is an intra cellular pathogen, cell mediated immunity, especially T cells play a crucial role in immune response. T cell function was primarily measured by their proliferative capacity against the crude and recombinant antigens as well as mitogen. Apart from that, IFN-γ is a key cytokine involved in the control of tuberculosis. So, cell proliferation and IFN-γ were used as the parameters to optimize the assay from long term culture condition (6 days). Long-term assays have advantages over short term assays, because during long term incubations, there will be an expansion of antigen specific IFN-γ secreting central memory T- cells. Therefore long-term assays are more sensitive to check diagnostic and vaccine potential of M. tuberculosis specific antigens .
As described by many authors , even though the conventional PBMC assays are specific in T cell functional analysis (since there is no influence of other whole blood components such as neutrophils and plasma), it requires large sample volume and there will be considerable cell loss during isolation.
The alternative T cell assay, WB assay is advantageous over PBMC assays; it requires small sample size, it is rapid and there is no need for cell separation. But the main disadvantage of this technique is that the number of cells cultured is neither known nor controlled. As such, variations in cytokine production between individuals, and in different physiological and pathological states, could represent either changes in the numbers of cytokine producing cells (e.g. T lymphocytes, monocytes) or an altered ability of those cells to produce cytokines, or both. In contrast, purified PBMC cultures include a precisely known number of cytokine-producing cells.
Thus, this study indicates that, although there is substantial inter-individual variation in ex vivo cytokine production, the production of any given cytokine by a particular individual is stable in the absence of changes in health and lifestyle factors, and that whole blood cultures can be used instead of purified PBMC cultures to measure a variety of cytokines.
In addition to describing the variation in cytokine production, the aim of this study was to identify whether whole blood cultures could be used to measure cytokine production rather than purified PBMCs. This might be an advantage in field work and/or where large numbers of blood samples need to be processed .
In this study, we employed two different whole blood dilution conditions i.e. 1:5 and 1:10 dilutions for T cell functional analysis in comparison to PBMC assays. For PBMC cultures, 10% AB serum which has been added provide nutrients that are equal to 1:10 diluted whole blood concentration. Thus, in this study we used the highest dilution factor i.e. 1:10.
Based on one way ANOVA results, we observed no statistically significant difference in lymphocyte proliferation in 1:5, 1:10 diluted WB and PBMC assays against all the three antigens and mitogen.
Whereas in the case of IFN-γ levels, there was no statistically significant difference in all the assays against recombinant protein and crude protein. But there was a significant increase (p = 0.0379) in IFN-γ levels in 1:10 diluted WB assay against mitogen, when compared to PBMC assay.
When compared to 1:5 diluted WB and PBMCs, stimulation index and IFN-γ levels were high in 1:10 diluted WB assays when stimulated with antigens and mitogen. Based on these results we concluded that 1:10 diluted WB assays are more promising and work better than both 1:5 diluted WB and PBMC assays.
In comparison with 1:5 diluted WB and PBMCs, availability of nutrients during long term incubation will be more in 1:10 diluted WB because of higher volume of RPMI 1640 medium, which would have helped to avoid cell crowding during the incubation. This might play an essential role in optimal T-cell activation and secretion of IFN-γ against the antigens and mitogen in 1:10 diluted WB assays.