Method
Detection of the interferon-gamma (IFN-γ) produced during the test. One commercial test (QFT) uses an enzyme-linked immunosorbent assay (ELISA) method to measure soluble cytokines. In contrast, the other method (T-Spot) employs an enzyme-linked immunospot assay (ELISPOT) method to stain the interferon-gamma in T cells. These newer-generation assays have improved specificity by decreasing false positives caused by interference from the BCG vaccine or nontuberculosis mycobacteria.
Note: Processing, testing, and interpretation steps are detailed and complex. Many public health and reference laboratories offer the test, but smaller medical laboratories may not have adequate staff or budget for this technically challenging, laborious, and expensive test.
Procedure
Note: This synopsis is intended to provide a basic understanding of the IGRA procedure; to provide detail is beyond the scope of this course. For more information, please consult individual vendor package inserts.
- Both methods require fresh blood to be drawn into a lithium heparin tube. (Note that sodium citrate is also acceptable for the ELISPOT method.)
- Blood samples must be mixed with antigens and controls.
- At this time, both IGRA methods utilize synthetic peptides (mycobacterial proteins) representing ESAT-6 and CFP-10; these proteins stimulate the patient cells in heparinized whole blood.
- ELISA and ELISPOT use 96-well microplates. A series of steps involves adding conjugate and substrate reagents and washing steps.
- The patient's T-cell lymphocytes release interferon-gamma (IFN-γ).
- Measurement of the IFN-γ is performed. In the QuantiFERON® method, the amount of IFN-γ released in response to the M. tuberculosis antigens and control substances is measured quantitatively, utilizing a standard curve. Numerical values include responses to two controls (nil and mitogen). In the SPOT® TB method, the number of interferon-gamma-producing cells (precipitate appearing as spots) is measured/counted. (Both quantitative and qualitative results should be reported along with interpretation.)
