How IGRA Works
IGRAs detect Mycobacterium tuberculosis infection by measuring interferon-gamma (IFN-γ) released from T lymphocytes when stimulated with specific M. tuberculosis antigens in a blood sample. 1
Basic Mechanism
The fundamental principle is straightforward: when blood from someone infected with M. tuberculosis is exposed to TB-specific antigens, their sensitized T cells recognize these antigens and release IFN-γ as part of the cell-mediated immune response. The amount of IFN-γ released is then quantified to determine if the person has been infected 2, 3.
Key Antigens Used
Modern IGRAs use highly specific M. tuberculosis proteins—primarily ESAT-6 (early secretory antigenic target-6) and CFP-10 (culture filtrate protein 10)—which are present in all M. tuberculosis strains but absent from BCG vaccine strains and most nontuberculous mycobacteria. 1 This is the critical advancement that makes IGRAs more specific than the tuberculin skin test (TST).
Important Caveat
ESAT-6 and CFP-10 are present in M. kansasii, M. szulgai, and M. marinum, which can cause false-positive results in individuals sensitized to these organisms 1.
Two Main IGRA Platforms
QuantiFERON-TB Gold In-Tube (QFT-GIT)
This ELISA-based test works as follows:
Blood collection: 1 mL of blood is collected into each of three specialized tubes containing:
- Antigen tube: Peptides representing ESAT-6, CFP-10, and TB7.7
- Negative control (Nil): Heparin alone
- Positive control: Phytohemaglutinin (to verify immune competence) 1
Incubation: Tubes are incubated for 16-24 hours at 37°C, allowing T cells to respond to antigens 1
Measurement: Plasma is separated and IFN-γ concentration is measured using a sensitive ELISA 1
Calculation: TB Response = IFN-γ in antigen tube minus IFN-γ in Nil tube 1
T-SPOT.TB
This ELISpot-based test uses a different approach:
Cell isolation: Peripheral blood mononuclear cells (PBMCs) are separated from whole blood 1
Stimulation: PBMCs are incubated with two separate peptide pools (one for ESAT-6, one for CFP-10) plus controls 1
Detection: Uses enzyme-linked immunospot assay to count individual IFN-γ-secreting T cells (appearing as "spots" in test wells) rather than measuring IFN-γ concentration 1, 2
Why This Works Better Than TST
The specificity advantage is substantial: Because ESAT-6 and CFP-10 are absent from BCG vaccine, IGRAs are not confounded by prior BCG vaccination—a major limitation of TST which uses purified protein derivative (PPD) containing antigens shared with BCG 1, 2.
The sensitivity consideration: Because ESAT-6 and CFP-10 are recognized by fewer T lymphocytes and stimulate less IFN-γ release compared to PPD, a more sensitive ELISA than earlier tests is required to detect the response 1.
Critical Requirements
IGRAs require viable, functional white blood cells to work properly 1. This means:
- Blood must be fresh and properly handled
- The patient must have adequate immune function
- Results may be indeterminate in severely immunocompromised individuals
The positive control tube is essential for verifying that the patient's immune system can respond to stimulation—if this fails, the test cannot be interpreted 1.
What IGRAs Cannot Do
Neither IGRAs nor TST can distinguish between latent TB infection and active TB disease, nor between recent and remote infection 3. They simply indicate that the immune system has been sensitized to M. tuberculosis at some point. Clinical correlation, chest imaging, and microbiological studies are required to exclude active disease 1.