Background: Interferon-gamma release assays (IGRAs) are blood tests used to measure the
amount of interferon-γ (IFN-γ) released by T lymphocytes after stimulation by antigens specific for the
diagnosis of latent tuberculosis infection. A mitogen serves as a positive control to assess the immune
function in IGRAs.
Methods: This in vitro study was conducted to evaluate IFN-γ production by human whole blood stimulated
with heat-treated and/or cation-supplemented phytohemagglutinin (PHA), concanavalin A (Con
A) and pokeweed mitogen (PWM), using QuantiFERON-TB Gold Kit ELISA tests.
Results: The optimal concentrations of PWM, Con A and PHA for IGRAs were 2 µg/mL, 5 µg/mL
and 10 µg/mL, respectively. The results showed that IFN-γ production in response to PWM was the
highest and PHA was the lowest amount. The median values of three mitogens were in the following
order: PWM≥Con A≥ positive control>>PHA-P>>negative control. PWM and PHA were heat stable,
while Con A was heat sensitive. The mitogen response of lymphocytes to untreated or heat-treated
PWM and heat-treated Con A was increased in 1 mM Ca2+-supplemented groups, whereas the response
to heat-treated PHA was decreased. Exposure to 1 mM Mg2+ had no effect on untreated or heat-treated
PWM, and a concentration of 1 mM Zn2+ inhibited the stimulation of un-treated PWM. We found that
calcium supplementation improved the PWM-induced production of IFN-γ.
Conclusion: Therefore, PWM is an appropriate mitogen for use as a positive control in IGRAs. It is a
potential indicator of cytokine production in the diagnostic as well as research settings, and calcium
supplementation improved stimulation.