Regional Medical Laboratory

QuantiFERON TB Gold Plus (QFT-Plus) is a 4th generation in vitro diagnostic test that uses a peptide cocktail containing ESAT-6 and CFP-10 proteins to stimulate cells, particularly lymphocytes. Numerous studies have demonstrated that the ESAT-6 and CFP-10 protein antigens stimulate Interferon-γ (INF- γ) responses in T cells from individuals infected with Mycobacterium tuberculosis, but generally not from uninfected or BCG-vaccinated individuals.

The QFT-Plus technology assesses cell-mediated responses through a quantitative measurement of Interferon-γ (IFN-γ). Detection of IFN-γ by an enzyme-linked immunosorbent assay (ELISA) is used to identify in vitro responses to these peptide antigens that are associated with M. tuberculosis (MTB) infection. The QFT-Plus aids in the diagnosis of active tuberculosis and in individuals with a LTBI. The QFT-Plus, an IGRA, can only be considered as helpful but insufficient for diagnosing M. tuberculosis complex infection in sick patients; however, a positive result can support the diagnosis of tuberculosis disease or LTBI. Also important to note is that some medical treatments (e.g. immunosuppressive drugs, chemotherapy, etc.) or conditions (e.g. lymphopenia, HIV infection/AIDS, viral or intracellular infections, etc.) can impair immune functionality and potentially reduce the IFN-γ responses.

In M. tuberculosis infections, CD4+ T cells play a critical role in immunological control through their secretion of the cytokines IFN-γ. Evidence now supports a role for CD8+ T cells participating in the host defense to M. tuberculosis by producing IFN-γ and other soluble factors, which activate the macrophages to suppress growth of M. tuberculosis, kill infected cells, or directly lyse intracellular M. tuberculosis organisms. IFN-γ producing MTB-specific CD8+ cells which have been detected in subjects with LTBI and with active TB. Moreover, ESAT-6 and CFP-10 specific CD8+ T lymphocytes are described as being more frequently detected in subjects with active TB disease versus LTBI and may be associated with a recent MTB exposure or infection onset.

QFT-Plus has two distinct TB antigen tubes:

1. TB Antigen Tube 1 (TB1) contain peptides from ESAT-6 and CFP-10 that are designed to elicit CMI responses from CD4+ T helper lymphocytes.
2. TB Antigen Tube 2 (TB2) contain peptides from ESAT-6 and CFP-10 that are designed to elicit CMI responses from CD4+ T helper lymphocytes; and an additional set of peptides targeted for the induction of CMI responses from CD8+ cytotoxic T lymphocytes. The results from tube 1 versus tube 2 may provide clues to distinguish a patient as to whether they have active tuberculosis or LTBI.

DETAILED INTERPRETATION AND SIGNIFICANCE OF THE TB GOLD QUANTIFERON PLUS

The interpretation of the TB Gold QuantiFERON Assay is very straight forward as long as it is Positive or Negative; however, an indeterminate result suggests an invalid result. Take into consideration that the assay is measuring the release of γ interferon due to the activation of the T helper cells (CD4+, tube 1) or the T helper (CD4+) and T cytotoxic cells (CD8+, tube 2) by Mycobacteria tuberculosis (MTB) antigens (ESAT-6 and CFP-10). Taking it step by step, first evaluate the Nil and the mitogen result and then the results of the TB1 and TB2.

Significance of the NIL result: The NIL indicates the presence of any residual interferon-γ found in the patient’s blood due to an infection with an organism requiring cell mediated immunity/T cell immunity (e.g. virus, fungus, intracellular bacteria, etc.). The NIL tube value indicates if the patient has an ongoing immune response which can cause a false-positive result. The NIL tube must be a value of no greater than 8.0 IU/mL.

Significance of the MITOGEN result: The mitogen control tube addresses the immune competence of the patient’s immune cells to produce interferon-γ. Thus a patient on immunosuppressive therapy (e.g. steroids), chemotherapy, or has a lymphopenia, or any reason for the patient’s lymphocytes to be unable to generate interferon-γ, will have very little if any response to the mitogen, indicating the lymphocytes’ inability to respond to an antigen.

A low response to a mitogen (<0.5 IU/ml) indicates an indeterminate result even when a blood sample also has a negative respond to the TB antigen. Indeterminate results from low mitogen values would not be expected to change upon immediate retesting. If a physician desires to retest a patient on immunosuppressive therapy or chemotherapy, wait 30-60 days before retesting. The mitogen tube also serves as a control for indicating long or improper transport, incorrect blood handling and incubation. If technical issues are suspected with the collection or handling of blood samples, repeat the entire QuantiFERON TB Gold Plus test with new blood specimens. The mitogen tube also acts as a false negative control. The mitogen tube must demonstrate an interferon-γ value of equal to or greater than 0.5 IU/ml higher than the value of the NIL tube.

Significance of the TB Antigen Results: QuantiFERON-TB Gold Plus (QFT-Plus) is a 4th generation in vitro diagnostic test using a peptide cocktail with ESAT-6 and CFP-10 proteins to stimulate cells, particularly T lymphocytes collected as whole blood in a 5 ml lithium heparin tube. The QFT-Plus technology assesses cell-mediated responses through a quantitative measurement of Interferon-γ (IFN-γ). Detection of IFN-γ by an enzyme-linked immunosorbent assay (ELISA) is used to identify in vitro responses to the peptide antigens. The QFT-Plus aids in the diagnosis of active tuberculosis but also in individuals with latent tuberculosis infection (LTBI), which is a noncommunicable asymptomatic condition, persisting in some, who might develop tuberculosis disease months or even years later. Distinguishing LTBI from active tuberculosis disease would be most helpful. Keep in mind that the QFT-Plus, an IGRA, is only helpful but insufficient for diagnosing M. tuberculosis complex infection in sick patients but a positive result can support the diagnosis of active tuberculosis disease or LTBI. Numerous studies have demonstrated that the ESAT-6 and CFP-10 protein antigens stimulate IFN-γ responses in T cells from individuals infected with M. tuberculosis but generally not from uninfected or BCG-vaccinated persons. It is important to note that some medical treatments (e.g. immunosuppressive therapy/drugs, chemotherapy, etc.) or conditions (e.g. lymphopenia, HIV infection/AIDS, etc.) can impair immune functionality and potentially reduce the IFN-γ responses.

In M. tuberculosis infection, CD4+ T cells play a critical role in immunological control through their secretion of the cytokine IFN-γ. Evidence now supports a role for CD8+ T cells participating in the host defense to M. tuberculosis by producing IFN-γ and other soluble factors, which activate the macrophages to suppress growth of M. tuberculosis, kill infected cells, or directly lyse intracellular M. tuberculosis organisms. IFN-γ producing MTB-specific CD8+ cells have been detected in subjects with LTBI and with active TB. Moreover, ESAT-6 and CFP-10 specific CD8+ T lymphocytes are described as being more frequently detected in subjects with active TB disease versus LTBI and may be associated with a recent MTB exposure or infection onset.

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For further assistance in interpretation of results, please contact Dr. Gerald Miller at 918-744-2553 ext. 15543 or the RML Client Services Department at 800-722-8077.

Follow the link below to review the current CDC Control and Prevention Guidelines for Diagnosis of Tuberculosis in Adults and Children:
https://www.idsociety.org/globalassets/idsa/practice-guidelines/official-american-thoracic-society.infectious-diseases-society-of-america.centers-for-disease-control-and-prevention-clinical-practice-guidelines-diagnosis-of-tuberculosis-in-adults-and-children.pdf