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Test code(s) 37737

What’s new (version 2): Q9: Does vaccination affect a T-SPOT.TB result? 

The T-SPOT®.TB test is an in vitro diagnostic test for the detection of effector T cells that respond to stimulation by Mycobacterium tuberculosis antigens ESAT-6 and CFP 10 by capturing interferon gamma (IFN- γ) in the vicinity of T cells in human whole blood collected in sodium citrate or sodium or lithium heparin.1 It is intended for use as an aid in the diagnosis of M tuberculosis infection.1

The T-SPOT.TB test is an indirect test for M tuberculosis infection (including disease) and is intended for use in conjunction with risk assessment, radiography, and other medical and diagnostic evaluations.1

Recommendations from the Centers for Disease Control and Prevention (CDC) state that2,3

  • “An interferon gamma release assay (IGRA) may be used in place of (but not in addition to) a tuberculin skin test (TST) in all situations in which CDC recommends TST as an aid in diagnosing adult M tuberculosis infections.”
  • “An IGRA is preferred for testing persons who have received Bacillus Calmette-Guérin (BCG) as a vaccine or for cancer therapy.”
  • “An IGRA or a TST may be used without preference to test recent contacts of persons known or suspected to have active TB with special considerations for follow-up testing. IGRAs offer the possibility of detecting M tuberculosis infection with greater specificity than with a TST. If IGRAs are to be used in contact investigations, negative results obtained prior to 8 weeks after the end of exposure typically should be confirmed by repeat testing 8 to 10 weeks after the end of exposure.” 

The T-SPOT.TB assay is a blood test for M tuberculosis infection that is based on measurement of a cell-mediated immune response.1 The peptide cocktail simulating the mycobacterial proteins ESAT-6 and CFP-10 stimulates the patient’s T cells in vitro to release IFN-γ, which is then measured using the enzyme-linked immunospot (ELISPOT) methodology.Peripheral blood mononuclear cells (PBMCs) are separated from a whole blood specimen, washed, and then counted before being added into the assay.1

Isolated PBMCs are exposed to a phytohemagglutinin control, nil control, and 2 separate panels of M tuberculosis–specific antigens. Enumerating the spots provides a measurement of the abundance of M tuberculosis–sensitive cells in the peripheral blood.1 This type of test is referred to as an IGRA.

Please refer to the package insert for full information on assay methodology.1

IGRA testing has a number of advantages over a TST, including

  • The physician’s practice does not need trained staff to generate the results.1
  • Accuracy is not affected by prior BCG vaccination.1,2
  • Requires only 1 patient visit (2-4 required for TST).1,2
  • Reduced staff time and avoidance of unnecessary follow-up, additional treatment, and treatment due to false-positive TST results caused by prior BCG vaccination.2

T-SPOT.TB test results are reported as positive, negative, or borderline (equivocal). For all samples, the test controls, positive and negative, must perform as expected.1 Test results are determined by enumerating the spots (captured interferon-gamma from individual T cells) in each of the patient’s 4 test wells (Positive Control, Nil Control, Panel A, Panel B).1 Qualitative results for the T-SPOT.TB test are interpreted by subtracting the spot count in the Nil control well from the spot count in each of the Panels, according to the following algorithm:

  • Positive: The test result is Positive if (Panel A-Nil) and/or (Panel B-Nil) spot count is ≥ 8.1

  • Negative: The test result is Negative if both (Panel A-Nil) and (Panel B-Nil) spot counts are ≤ 4, including values <0.1

  • Borderline (equivocal): Results where the highest of the Panel A or Panel B spot count is 5, 6, or 7 spots are considered Borderline (equivocal); retesting by collecting another patient specimen is recommended.1

If the result is still Borderline (equivocal) on retesting with another specimen, then other diagnostic tests and/or epidemiologic information should be used to help determine the tuberculosis (TB) infection status of the patient.1

Please refer to the package insert for full information on result reporting/interpretation.1

Conversion: with respect to TB antigens, conversion is the point at which interferon gamma becomes detectable above the established threshold or cutoff from a previous negative or unknown result (negative to positive).1,2,4 In a study of over 16,000 healthcare workers across 19 US hospitals, the mean conversion rate was 0.8% (range, 0.0%-2.5%).4

Conversion rates correlate with geographic incidence of TB and/or known TB risk factors. For patients and healthcare workers with weak-positive results in low-risk settings, CDC guidelines recommend retesting with an equivalent or other test while considering the full clinical picture and risk/exposure assessment.4

Reversion: the opposite of conversion—a result that goes from above the established cutoff to below the cutoff (positive to a negative). In a study of over 16,000 healthcare workers across 19 US hospitals, the mean reversion rate was 17.6%. Hospitals using T-SPOT.TB to evaluate only high-risk healthcare workers trended lower in reversion rate (mean 13.9%) when compared to hospitals evaluating all healthcare workers (mean 20.7%).4

Results are considered invalid for any of the following reasons1

  • Nil well develops >10 spots.

  • Mitogen well develops <20 spots (unless Panel A-Nil and/or Panel B-Nil are Positive or Borderline).

  • High background staining hinders discrimination of the spots from the background in the microwells.

Invalid results can be due to 1 or more of the following reasons1

  • Operator/testing issues

  • Medium contamination

  • Improper specimen handling

  • Improper separation of PBMCs

  • Patient health (ie, low PBMC count leading to inadequate response to mitogen)

  • Inadequate response to mitogen without known cause

Repeat testing by collecting another specimen is recommended for invalid results.1

No. The T-SPOT.TB test has not been evaluated in individuals who have received >1 month of anti-TB therapy.1

Yes, a prior TST can boost IGRA results.2,5 However, boosting does not occur in uninfected individuals, because they do not have an established immune response to TB.2 Furthermore, the TST, despite its in vivo application, does not cause sensitization or establish a cell-mediated response.2,5 Therefore, it cannot cause subsequent boosting among persons without prior TB infection.2,5

The implications of a boosted response are unknown. However, a boosted response is thought to reflect the induction of a weak memory response from multiple points of stimulation.2,5 Boosting is a common phenomenon when a TST is repeated. Each TST can boost subsequent TST responses, due to remote TB infection, as well as infection with non-tuberculous mycobacteria or BCG vaccination.2,5

The CDC previously recommended either the TST or IGRA be performed >4 weeks after completion of COVID-19 vaccination, to minimize potential interference between vaccination and TB testing. This recommendation reflected an abundance of caution during a period when these vaccines were new. These tests may now be administered without regard to timing of COVID-19 vaccination and can even be administered during the same encounter as COVID-19 vaccination.6

Similarly, COVID-19 vaccination should not be delayed because of testing for TB infection.6

Live-virus vaccines: Live-virus vaccines might affect IGRA test results.7 However, the effect of live-virus vaccination on IGRAs has not been studied.7 Until additional information is available, IGRA testing in the context of live virus vaccine administration should be done as follows7:

  • Either on the same day as vaccination with live-virus vaccine or 4-6 weeks after the administration of the live-virus vaccine
  • At least 1 month after smallpox vaccination

Yes. An IGRA can be used for children 2 years of age or older. It is preferred for children who have had the BCG vaccine or are unlikely to return for a TST to be read.

For children <2 years of age, the TST is the preferred test to detect TB infection.8

T-SPOT.TB specimen requirements for children are as follows1:

  • <2 years: 2 mL lithium heparin
  • 2-10 years: 4 mL lithium heparin
  • ≥10 years: 6 mL lithium heparin


  1. T-SPOT.TB. Package insert. Oxford Immunotec Inc; 2017. Accessed September 20, 2022.
  2. Lewinsohn DM, Leonard MK, LoBue PA, et al. 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. Clin Infect Dis. 2017;64(2):e1-e33. doi:10.1093/cid/ciw694
  3. Mazurek GH, Jereb J, Vernon A, et al. Updated guidelines for using interferon gamma release assays to detect Mycobacterium tuberculosis infection—United States, 2010. MMWR Recomm Rep. 2010;59(RR05):1-25.
  4. King TC, Upfal M, Gottlieb A, et al. T-SPOT.TB interferon-γ release assay performance in healthcare worker screening at nineteen US hospitals. Am J Resp Crit Care Med. 2015;192(3):367-373. doi:10.1164/rccm.201501-0199OC
  5. van Zyl-Smit RN, Zwerling A, Dheda K, et al. Within-subject variability of interferon-g assay results for tuberculosis and boosting effect of tuberculin skin testing: a systematic review. PLoS One. 2009;4(12):e8517. doi:10.1371/journal.pone.0008517
  6. Chorba T. Dear colleague letters: COVID mRNA vaccines, TST, and IGRA – follow-up. Centers for Disease Control and Prevention. Published August 31, 2021. Accessed August 24, 2022.
  7. TB elimination: interferon-gamma release assays (IGRAs) – blood tests for TB infection. Centers for Disease Control and Prevention. Published November 2011. Accessed Sept. 20, 2022.
  8. American Academy of Pediatrics. Tuberculosis. In: Kimberlin DW, Brady MT, Jackson MA, et al, eds. Red Book: 2018 Report of the Committee on Infectious Diseases. 31st ed. American Academy of Pediatrics; 2018:829-53.                                                       


This FAQ is provided for informational purposes only and is not intended as medical advice. A clinician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.

Document FAQS.215 Version: 2

Version 2: Effective 10/24/2022 to present

Version 1: Effective 03/04/2021 to 10/24/2022
Version 0: Effective 12/02/2019 to 03/04/2021