Interferon-Gamma Release Assay (IGRA): Principle, Procedure, and Interpretation

A previously BCG-vaccinated nurse has a 12 mm tuberculin skin test after a ward exposure. Is that induration from the vaccine or from real infection? This is the question the IGRA was designed to answer, and reading its result correctly is the difference between clearing a healthcare worker and starting unnecessary treatment.

What an IGRA measures

An interferon-gamma release assay is a blood test that detects a person's immune memory of Mycobacterium tuberculosis. When T cells that have previously met M. tuberculosis are re-exposed to its antigens in the tube, they release interferon-gamma (IFN-γ). The assay measures that release. A response means the immune system has seen the organism; it does not, on its own, tell you whether the infection is latent or active.

Like the tuberculin skin test, an IGRA is an indirect test. It is positive in both latent tuberculosis infection and active tuberculosis, so any positive result requires clinical evaluation for active disease before it is acted on.

Why the IGRA was developed

The tuberculin skin test has two long-standing weaknesses. It cross-reacts with the BCG vaccine, so a vaccinated person can have a positive skin test with no true infection, and it cross-reacts with some environmental non-tuberculous mycobacteria. It also requires the patient to return in 48 to 72 hours for the reading, and the induration measurement is operator-dependent.

The IGRA addresses the specificity problem directly. It uses antigens that are specific to the M. tuberculosis complex, mainly Early Secretory Antigen Target 6 (ESAT-6) and Culture Filtrate Protein 10 (CFP-10). These antigens are absent from the BCG vaccine strain and from most environmental mycobacteria. As a result, BCG vaccination does not cause a positive IGRA. The test is also completed on a single blood draw with no return visit.

The two IGRA formats

Two formats are in routine use, and they differ in what they measure.

1. QuantiFERON-TB Gold (current version: QuantiFERON-TB Gold Plus) measures the concentration of IFN-γ released into plasma, reported in international units per milliliter (IU/mL), using an ELISA readout. Whole blood is collected directly into antigen-coated tubes.
2. T-SPOT.TB counts the number of individual T cells that release IFN-γ, using an ELISPOT method. Peripheral blood mononuclear cells are separated first, then exposed to the antigens, and the result is reported as the number of spot-forming units.

Both use the same ESAT-6 and CFP-10 antigen principle. QuantiFERON asks "how much IFN-γ," T-SPOT asks "how many responding cells."

Procedure

The value of an IGRA comes from its built-in controls, so the tube set matters more than the pipetting. For QuantiFERON-TB Gold Plus, blood is drawn into four tubes:

1. The Nil tube contains no antigen. It measures the background IFN-γ already present in the blood, which is subtracted from the antigen result. A high Nil value signals a problem.
2. The two TB antigen tubes (TB1 and TB2) contain the ESAT-6 and CFP-10 peptides. TB1 is designed to elicit a CD4 T-cell response; TB2 adds antigens that also elicit a CD8 T-cell response, which can matter in active and in some immunocompromised patients.
3. The Mitogen tube contains a non-specific T-cell stimulant (phytohemagglutinin). It is a positive control: if the person's T cells are functional, this tube should always produce IFN-γ. A failed mitogen response tells you the immune cells could not respond at all, which is how an indeterminate result is identified rather than misreported as negative.

Tubes are incubated at 37 °C, plasma is separated, and IFN-γ is measured by ELISA. The result is calculated from the antigen tubes after subtracting the Nil value.

Interpretation

This is the section that matters most, because the syllabus sets IGRA at the "interpret" bar, and because the interpretation queries are where the recoverable traffic sits.

1. Positive. The IFN-γ released in a TB antigen tube, minus the Nil, exceeds the assay cutoff. This means M. tuberculosis infection is likely. It does not distinguish latent from active disease. A positive result always triggers clinical and radiological evaluation for active tuberculosis.
2. Negative. The TB antigen response is below the cutoff and the Mitogen (positive control) responded normally. This makes M. tuberculosis infection unlikely, but it does not exclude it, particularly early after exposure or in significant immunosuppression.
3. Indeterminate. This is the result students most often misread, and the one the controls exist to catch. It arises in one of two ways. Either the Mitogen tube failed to produce IFN-γ, meaning the T cells could not respond even to a strong non-specific stimulus, which points to immunosuppression, lymphopenia, or a processing/handling error. Or the Nil tube IFN-γ was too high, meaning background reactivity was so elevated that a true antigen response could not be distinguished. An indeterminate result is not a weak positive or a weak negative; it means the test cannot be interpreted and usually needs to be repeated, with attention to sample handling and to the patient's immune status.

A practical point that follows directly from the control logic: an indeterminate result in an apparently healthy person is more often a handling problem (delayed incubation, incorrect tube filling, poor mixing) than a true immune finding. Check the pre-analytical steps before concluding anything about the patient.

IGRA versus tuberculin skin test: a point-by-point comparison

Both the interferon-gamma release assay and the tuberculin skin test answer the same question: has this person's immune system met Mycobacterium tuberculosis? Neither answers the question that usually matters more, which is whether the infection is active. The two tests reach the same kind of answer by different routes, and the differences between those routes decide which test fits a given situation.

The tuberculin skin test injects purified protein derivative into the skin and measures the delayed immune reaction as induration at 48 to 72 hours. It happens in the body, in the skin, and it is read by eye. The IGRA takes blood, exposes the person's T cells to M. tuberculosis-specific antigens in a tube, and measures the interferon-gamma they release. It happens in the laboratory and is read by an instrument.

Three differences follow from that, and they are the ones worth remembering.

Specificity and BCG. The tuberculin skin test uses PPD, a crude mixture of many mycobacterial proteins, some of which are shared with the BCG vaccine strain and with environmental mycobacteria. A person vaccinated with BCG can therefore have a positive skin test with no true infection. The IGRA uses ESAT-6 and CFP-10, antigens that are absent from BCG and from most environmental mycobacteria, so BCG vaccination does not cause a positive IGRA. This is the single most important difference and the main reason IGRA was developed.

Logistics and the return visit. The skin test requires two visits: one to place it, one to read it 48 to 72 hours later. A person who does not return cannot be tested. The IGRA needs a single blood draw with no return visit, which matters for populations unlikely to come back for a reading. Against that, the IGRA requires prompt laboratory processing; a delay in incubating the sample degrades the result, which the skin test does not have to worry about.

Reading and subjectivity. The skin test induration is measured with a ruler and depends on the reader's technique and judgment, and on the patient's own reporting if the reader is not the same person who placed it. The IGRA readout is quantitative and instrument-based, with built-in positive and negative controls (the Mitogen and Nil tubes) that flag an uninterpretable result as indeterminate rather than letting it pass as a false negative.

What the two share is just as important as where they differ. Neither test distinguishes latent infection from active disease. Both are positive in latent and active tuberculosis alike, and a positive result from either one requires evaluation for active disease before it is acted on. Neither should be used alone to diagnose active tuberculosis, which rests on direct methods: microscopy, culture, and nucleic acid amplification.

When to choose which. The IGRA is preferred when the person has had BCG vaccination, because it is not confounded by it, and when a return visit is unlikely, because it needs only one draw. The tuberculin skin test remains a reasonable choice where IGRA is unavailable or unaffordable, in settings without prompt laboratory access, and in serial testing programs where its behavior over time is well understood. In young children, guidelines in many settings still favor or combine the skin test, where IGRA evidence is more limited.

IGRA vs tuberculin skin test in one table

How to remember the difference

One antigen fact carries most of the comparison: PPD is a crowd, ESAT-6 and CFP-10 are a shortlist. PPD shares proteins with BCG, so the skin test reacts to the vaccine; the IGRA's shortlist antigens are not in BCG, so it does not. If you remember only that, you can reconstruct the specificity difference, the false-positive-with-BCG point, and the reason the IGRA exists.

For logistics: skin test needs the patient to come back, IGRA needs the sample to move fast. Each test has one weak link in its workflow, and they are opposite kinds.

Limitations

An IGRA cannot separate latent infection from active tuberculosis, so it cannot be used to rule active disease in or out. It can give false results in profound immunosuppression, where T cells may not respond even when infection is present. It is more expensive and more laboratory-intensive than the skin test and requires prompt sample processing, since delays in incubation degrade the result. It is not recommended as the sole test in young children in many guidelines, where evidence is more limited.

How to remember

The three tubes tell the whole interpretation story, so anchor the memory there rather than on a list of results:

• Nil = background. If Nil is high, the test drowns in noise, indeterminate.
• TB antigen = the question. High means infection likely.
• Mitogen = "can these cells even respond?" If Mitogen is flat, the cells are not answering, indeterminate, not negative.

The single fact that separates IGRA from the skin test: ESAT-6 and CFP-10 are not in BCG. That one antigen fact is why BCG does not cause a positive IGRA, and it is the reason the test exists.

Key exam facts

Where students get confused

"Indeterminate means the patient is borderline." No. Indeterminate means the assay could not be interpreted, almost always because the Mitogen control failed or the Nil background was too high. It is a statement about the test, not a statement about the patient's infection status. The first response is to check sample handling and repeat, not to treat.

"A positive IGRA means active TB." No. IGRA is positive in latent infection and in active disease equally. It cannot tell them apart. A positive result starts a work-up for active disease; it does not diagnose it.

"IGRA is affected by BCG like the skin test." The opposite is the whole point of the test. ESAT-6 and CFP-10 are not present in the BCG strain, so vaccination does not produce a positive IGRA.

"A negative IGRA rules out TB." It makes infection unlikely but does not exclude it, especially soon after exposure or in immunosuppression. Direct tests are needed to address active disease.

References

• Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.
• Procop GW, et al. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017.
• Leber AL, editor. Clinical Microbiology Procedures Handbook. 4th ed. Washington, DC: ASM Press; 2016. doi:10.1128/9781683670438.CMPH
• World Health Organization. WHO consolidated guidelines on tuberculosis. Module 3: Diagnosis, tests for tuberculosis infection. Geneva: WHO. 2025