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Test code(s) 37737

Question 1. What is the T-SPOT®.TB test?

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

Question 2. How does the T-SPOT.TB test work?

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.1

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

Question 3. What are the advantages of T-SPOT.TB, or interferon gamma releasing assay (IGRA), over tuberculin skin test (TST)?

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

  • Results are not subject to reader bias.1

  • Accuracy is not affected by prior bacille Calmette-Guérin (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.2

Question 4. How do you interpret the results of T-SPOT.TB?

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

Question 5. What are conversion and reversion?

Conversion: With respect to TB antigens, conversion is the point at which IFN-γ becomes detectable above the established threshold or cutoff from a previous negative or unknown result (negative to positive).1-3 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%).3

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, Centers for Disease Control and Prevention (CDC) guidelines recommend retesting with an equivalent or other test while considering the full clinical picture and risk/exposure assessment.3

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%).3

Question 6. What causes an invalid result?

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

Question 7. Can T-SPOT.TB testing be used to monitor TB therapy?

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

Question 8. Does prior TST affect a T-SPOT.TB result?

Yes, a prior TST can boost IGRA results.2,4 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.2,4 Therefore, it cannot cause subsequent boosting among persons without prior TB infection.2,4 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.2,4 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 vaccination with BCG.2,4

Question 9. Does vaccination affect a T-SPOT.TB result?

Vaccination with a live-virus vaccine

The effect of live-virus vaccination on T-SPOT.TB has not been studied. However, it is advisable that the T-SPOT.TB test specimen be drawn either5

  • The same day as administration of a live-virus vaccine, or

  • Four to six weeks after the administration of a live-virus vaccine

COVID-19 mRNA vaccine

Both the Pfizer-BioNTech and Moderna COVID-19 vaccines are inactive vaccines and do not interfere with TB test results.6 There is no immunologic reason to believe that a blood draw for T-SPOT.TB testing would affect the safety or effectiveness of the mRNA COVID-19 vaccines.6,7

CDC recommendations for TB testing before and after COVID-19 mRNA vaccination are as follows:

Question 10. Can IGRAs be used in children?

Yes. An IGRA can be used for children 2 years of age or older. It is preferred in 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; 2013.
  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. 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
  4. 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
  5. TB elimination: interferon-gamma release assays (IGRAs) – blood tests for TB infection. Centers for Disease Control and Prevention. Accessed March 2, 2021. https://www.cdc.gov/tb/publications/factsheets/testing/IGRA.pdf
  6. Dear colleague letters. Centers for Disease Control and Prevention. Reviewed January 7, 2021. Accessed March 2, 2021. https://www.cdc.gov/tb/publications/letters/covid19-mrna.html
  7. Sax, PE. COVID-19 – vaccine frequently asked questions. New England Journal of Medicine. Updated February 2021. Accessed February 16, 2021. https://www.nejm.org/covid-vaccine/faq
  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.
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Version 1: Effective 03/04/2021 to present
Version 0: Effective 12/02/2019 to 03/04/2021