T-SPOT^®.TB

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 that^2,3

• “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.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 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