Immune Response to Mycobacterial Infection: Lessons from Flow Cytometry
Table 1
Comparison of tuberculin skin test (TST), interferon gamma release assay (IGRA), and flow cytometry assay.
TST
IGRAs
Flow Cytometry
Method
A valid TST requires proper administration by the Mantoux method with intradermal injection of 0.1 mL of tuberculin PPD into the volar surface of the forearm
A single specimen of peripheral blood is drawn and incubated in vitro overnight with Mtb-specific antigens
IntacT-cells and their constituent components are tagged with fluorescently conjugated monoclonal antibodies and/or stained with fluorescent reagents and then analyzed individually. Cells and the fluorescent molecules in/on each cell are excited by passing through the laser light at speeds exceeding 70,000 cells per second. Each cell passing through the beam also scatters light providing an indication of cell shape and size
Characteristics
A delayed-type hypersensitivity to intradermal injection of tuberculin-PPD testing for cell-mediated immunity against Mtb
The QFT-GIT measures the amount of INF- whereas the T-Spot determines the number of cells producing INF-; therefore they provide limited information regarding the complete phenotype of cells engaged in cytokine production or the kinetics of this response [19]
Allows analysis of multiple parameters per cell and accurately locates the pool of immunological effector cells responsible for cytokine production [19]
Yes (in 18 studies involving 1,169,105 subjects, the absolute prevalence of false-positive TST from NTM cross-reactivity ranged from 0.1% to 2.3% in different regions [36])
Yes, but less extensive than TST (ESAT-6 and CFP-10 are present in MTM M. kansasii, M. szulgai, and M. marinum, and sensitization to these organisms might cause false-positive IGRA results [3])
No (evidence suggests that flow cytometry might actually discriminate between infection with Mtb or NTM [37])
No (however, careful interpretation of true rather than artifactual (nonspecific) reactions is essential when the number of spots is counted in T-spot)
Expertise is required for correct and reproducible gating
Settings
Errors in intradermal administration, interpretation, and interreader variability
Errors in collecting or transporting blood specimens or in running and interpreting the assay can decrease the accuracy of IGRAs
Due to the need for technical expertise and expensive equipment, it is recommended that this assay be done only in a reference laboratory setting
NTM: nontuberculous mycobacteria; BCG: Bacille de Calmette et Guérin.