Antigen-induced cytokine and chemokine release test for tuberculosis infection using adsorption of stimulated whole blood on filter paper and multiplex analysis

Abstract

Background: In vitro stimulation of whole blood or isolated blood cells with specific antigens is used for several purposes. Immediately following incubation with antigens, samples have to be centrifuged to stop the reactions by remaining cells and the supernatant refrigerated or analysed directly to preserve the analytes of interest, which makes samples difficult to prepare outside laboratories. We have tested whether spotting whole blood on filter paper after activation can be used in one of the tests for Mycobacterium tuberculosis infection (MTI), the QuantiFERON®-TB Gold In Tube test (QFT), where the spotting technique can make it suitable for use in locations without facilities like a centrifuge and a refrigerator. Materials and methods: Samples from 22 individuals undergoing screening for MTI and 10 healthy controls were incubated, centrifuged and IFN-γ measured by Enzyme-linked immunosorbent assay (ELISA), as described in the kit insert. In parallel, activated blood was spotted on filter paper (Schleicher & Schuell) and dried. The dried blood spot samples were analysed for 21 inflammatory markers with an in-house assay based on Luminex technology. Results: Our multiplex measurements of inflammatory markers in samples from suspected MTI patients confirmed the IFN-γ findings in the QFT. IL-2, GM-CSF, IL-5, and IL-1β were also found as useful markers for MTI. We were not able to distinguish between active tuberculosis and latent MTI. Conclusion: Applying blood on filter paper after incubation makes in vitro stimulation tests feasible in locations where heat and electricity is unavailable.

Key Words::

• Immunologic stimulation
• antigens
• cytokines
• in-vitro
• Mycobacterium tuberculosis

Acknowledgements

We would like to thank Malene Billsten Zent, Karin Skaarup, Christina Lis Bonde, Lisette Jensen, and Jannie Hein Pedersen for skillful technical assistance. Further, we are very grateful to the volunteer blood donors.

Declaration of interest: The authors declare that they have no conflict of interest. The authors alone are responsible for the content and writing of the paper.