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-𝛾 whereas the T-Spot determines the number of cells producing INF-𝛾; 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]
Sensitivity (%)	0.65	QFT-G-IT 0.80; T-SPOT.TB 0.81 [8]	See Table 2
Specificity (%)	0.75	QFT-G-IT 0.79; T-SPOT.TB 0.59 [8]	See Table 2
T-cell populations detected	Primary central memory T-cells [6]	Primary effector memory T-cells [6]	All T-cell populations can be detected
TB stage	More likely to identify persons with longstanding cellular immune responses to these antigens [6]	More likely to identify persons who have recently been infected with M.  tuberculosis, a group at particularly high risk for progression to disease [6]	Promising tool for the identification of all stages of TB infection
Immunosuppression	Compromised performance [9, 33]	Compromised performance [9, 10]	Unaffected performance [31–33]
Cross-reactivity with BCG	Yes [3, 36]	No	No [31]
Cross reactivity with NTM	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])
“Booster” phenomenon	Yes	No	No
Patient visits	2	1	1
Processing time		Within 16 (QFT-GIT) to 30 (T-spot) hours	45 minutes–1 hour (average)
Time to results	48–72 hours	Within 16 (QFT-GIT) to 30 (T-spot) hours	Within 24 hours (~8 h) [35]
Interreader variability	Yes	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.