IGRA (Interferon-Gamma Release Assay) Test-Introduction, Principle, Test Requirement, Procedure, Result-Interpretation, and Keynotes

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Introduction

An Interferon-Gamma Release Assay (IGRA) is a modern blood test used to identify if a person is infected with Mycobacterium tuberculosis. It is primarily used to detect latent TB infection (LTBI) and to aid in the diagnosis of active tuberculosis disease. 

Principle

The test is based on the fact that T-lymphocytes from people previously infected with TB will release interferon-gamma (IFN) when they encounter specific TB antigens. 

  • Antigens Used: The tests use synthetic peptides that simulate specific proteins (ESAT-6 and CFP-10) found in M. tuberculosis.
  • Specificity: These antigens are absent from the BCG vaccine and most non-tuberculous mycobacteria, which prevents false-positive results in BCG-vaccinated individuals.
  • Measurement: The test measures the concentration of released IFN- or the number of T-cells secreting it. 

Test Requirements

  • Sample Type: Whole blood collected by venipuncture.
  • Fasting: Generally not required; patients can eat and drink normally before the test.
  • Timing: Blood must be processed or incubated within a strict timeframe (often within 8 to 16 hours of collection) to ensure cell viability.
  • Handling: Samples must be maintained at room temperature; they should not be refrigerated or frozen before incubation. 

Procedure

There are two main types of IGRA tests with slightly different laboratory procedures: 

Feature QuantiFERON-TB Gold Plus (QFT-Plus)T-SPOT.TB
MethodEnzyme-Linked Immunosorbent Assay (ELISA)Enzyme-Linked Immunospot (ELISPOT)
Collection4 specialized tubes (Nil, TB1, TB2, Mitogen)Standard lithium-heparin (green-top) tube
Lab StepMeasures the total concentration of IFN-  in plasmaSeparates and counts individual IFN-  secreting cells
Incubation16–24 hours at 37°C16–20 hours at 37°C

Result Interpretation

Results are calculated based on the response to TB antigens compared to negative (Nil) and positive (Mitogen) controls. 

  • Positive: Suggests that M. tuberculosis infection is likely. Further medical evaluation (e.g., chest X-ray, sputum culture) is required to rule out active disease.
  • Negative: TB infection is unlikely.
  • Indeterminate / Borderline: The test did not provide a clear result. This can be due to a patient’s weakened immune system or technical errors during blood handling. 

Keynotes

  • BCG Immunity: Unlike the skin test (TST), IGRA results are unaffected by prior BCG vaccination.
  • Single Visit: Only requires one clinic visit for the blood draw, whereas the skin test requires a second visit to “read” the result.
  • Limitations: IGRA cannot distinguish between latent infection and active TB disease. It is also not useful for monitoring a patient’s response to TB treatment.
  • Clinical Context: Results must always be interpreted alongside other clinical findings, such as risk factors and symptoms. 

Further Readings

  1. https://www.cdc.gov/tb/hcp/testing-diagnosis/interferon-gamma-release-assay.html
  2. https://www.questdiagnostics.com/healthcare-professionals/clinical-education-center/faq/faq204
  3. https://wwwn.cdc.gov/nchs/data/nhanes/public/2019/labmethods/TB-K-R-MET-508.pdf
  4. https://www.qiagen.com/us/tb-testing/what-is-quantiferon/how-does-qft-work
  5. https://www.health.nsw.gov.au/Infectious/tuberculosis/Pages/interferon-gamma-release-assay-test.aspx
  6. https://testdirectory.questdiagnostics.com/test/test-detail/37737/t-spottb?cc=MASTER
  7. https://www.labcorp.com/tests/182879/quantiferon-tb-gold-plus
  8. https://pmc.ncbi.nlm.nih.gov/articles/PMC3910908
  9. https://www.sgh.com.sg/our-specialties/pathology/pathology-lab-services/t-spot-tb-test
  10. https://www.cdc.gov/tb/webcourses/tb101/page202.html
  11. https://www.maven-help.maventrainingsite.com/pdf
  12. http://www.bovinetb.info/docs/igra-testing-for-tb-questions-and-answers.pdf
  13. https://pmc.ncbi.nlm.nih.gov/articles/PMC5457919

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