Acta Trop. 2013 Aug;127(2):75-81.

A whole blood assay as a simple, broad assessment of cytokines and chemokines to evaluate human immune responses to Mycobacterium tuberculosis antigens.

Silva D, Ponte CG, Hacker MA, Antas PR.

Laboratório de Imunologia Clínica, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, Brazil.


In vitro stimulation of whole blood or isolated peripheral blood cells with specific antigens is used for several purposes. We sought to identify a reliable, reproducible, fast and feasible in vitro method to assess human cellular immune responses to Mycobacterium tuberculosis. In contrast to peripheral blood mononuclear cell (PBMC) culture, a whole blood assay (WBA) provides a more physiological environment, which may provide a broader assessment of serum biomarker, biosignature profiles. Twenty-three asymptomatic individuals with M. tuberculosis infection were recruited. Total cells from the WBA (diluted 1:3 in completed RPMI) and PBMC (2×10(5)cells/ml) plus M. tuberculosis Ag85A, Ag85B, ESAT-6 and Mycobacterium bovis 65kDa were characterized by flow cytometry, then added in 96-well plates and on day 5 plasma and supernatants were harvested for detection of 17 cytokines by a Luminex array system. There was agreement between PBMC and WBA for IL-2, IL-5, IL-6, IL-7, IL-13, IFN-γ, TNF-α, MCP-1 and MIP-1β. There was evidence toward higher IL-10 (p≤0.049) and G-CSF (p≤0.012) plasma production, and higher IL-1β (p≤0.048), IL-4 (p≤0.044), IL-12p70 (p≤0.006), IL-17 (p≤0.002) and GM-CSF (p≤0.049) production for PBMC vs. WBA. Both methods provided virtually no reaction to the internal, negative control. Due to technical issues linked to data out of range, IL-8 data were not considered. These results suggest that, depending on the method employed, PBMC and/or WBA techniques provide fine conditions for the model proposed and thus whole blood cultures are well-suited low-cost proxy-measures during search for serum biomarkers. Copyright © 2013 Elsevier B.V.

PMID: 23571106



In years to come, there are potential roles for the study of cytokines in health and disease. Typical studies employing cell isolation procedures are restricted in their scope since they do not comprise all cell-cell or cell-protein interactions that physiologically occur in vivo. On the other hand, whole-blood assay (WBA) serves as a valuable connection between using normal individuals and PBMC. WBA may be used to study the immune response in an attempt to find out how cytokine regulation may take place in either patients or healthy volunteers. The WBA offers several advantages over conventional PBMC. (1) Very small volumes of venous blood are necessary to set up the assay; this can be extremely important for studies involving children or other immune impaired individuals; (2) The integral plasma present in the WBA is a much more physiologic culture medium than even the most complete tissue culture formulation; (3) The cell isolation procedures, which requires equipment and reagents that may not be available in remote settings, might activate leucocytes resulting in high background levels of cytokines. That makes WBA is broadly available even in regions in which severe, chronic diseases are present whereas sophisticated laboratories are not. For instance, the WBA usually has the most direct use in sepsis, obviously because of the circulation, however it has been employed to investigate other sorts of infections, including mycobacteria in low-income countries as well. Thus, the more interesting feature of the WBA is its use compared to PBMC, and thus our study demonstrates for the first time different aspects depending on the experimental conditions. For instance, a striking fold-increase for IL-17 and G-CSF is more evident upon specific Mycobacterium tuberculosis antigenic stimulation, when comparing PBMC vs. WBA, respectively (Table). In another example, only 5 out of 17 cytokines screened performed better in the PBMC system: IL-1β, IL-4, IL-12p70, IL-17 and GM-CSF. In WBA, it is quite difficult to determine which cells account for the production of a specific cytokine in a heterogeneous cell population, since PMN neutrophils correspond to the absolute numbers of major leucocytes in the circulation. In addition, cells from the site of inflammation may act differently than those in the circulation. Therefore, the WBA mimics the human blood condition, it is inexpensive and easy to perform. Taken together, our results suggested that, depending on the method employed, WBA may provide better conditions for the study of leukocyte cytokines/chemokines production and assessment of serum biomarkers or biosignature profiles in health and disease.


Table 1. Marked fold-increase ratio for cytokines between the 2 culture methods employed.


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