T-SPOT^®TB

T-SPOT^®TB 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 T-SPOT^®TB 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

For more information see the T-SPOT^®TB package insert.¹ 

Like the tuberculin skin test (TST), the interferon gamma release assay (IGRA) reflects the cellular immune response to Mycobacterium tuberculosis (Mtb) antigens by measuring the production of interferon-gamma (IFN-γ).¹ For the T-SPOT^®TB assay, blood is drawn into a heparin (green top) tube from which peripheral blood mononuclear cells (PBMCs) are extracted.¹ A required number of isolated PBMCs for each patient are added to the assay plate which has been coated with antibodies to capture soluble IFN-γ, and then stimulated with bacterial peptides derived from ESAT-6 and CFP 10 antigens.¹

After an overnight incubation, the plate is processed for visualization of captured IFN-γ, which appears as spots (ELISPOT assay).¹ These spots are regarded as an indication that effector T cells, which have been previously exposed to Mtb, generated a secondary response to the antigens in the assay plate.¹ In parallel to the Mtb peptide antigens, each patient’s PBMCs are incubated with no antigen (negative control) and a nontuberculous mitogen (positive control) to correct for background response and ascertain viable cellular function, respectively.¹

According to CDC, the IGRA tests serve the same purpose as the tuberculin skin test, but they have functional and diagnostic differences.^2-5

IGRA Advantages:

• Requires only one patient visit to do the test.
• Does not cause booster phenomenon (unlike the TB skin test)
• Not subject to biases and errors associated with TB skin placement and reading.
• Results can be available within 24 hours.
• Unaffected by the bacille Calmette-Guérin (BCG) TB vaccine and most nontuberculosis mycobacteria

IGRA Disadvantages:

• Blood samples must be processed within 2 days of collection due to the limited stability of live cells.
• Errors in collecting or transporting blood specimens or in running and interpreting the test can decrease the accuracy of TB blood tests.
• It may take several days for results to be available, depending on the laboratory and clinic.
• Tests may be more expensive than TB skin tests.

Test results for T-SPOT^®TB are interpreted by subtracting the spot count (captured interferon-gamma from individual T cells) in the NIL [negative] control well from the spot count in each of 3 other patient test wells [Positive Control, Panel A, Panel B].¹

• The test result is Positive if (Panel A-Nil) and/or (Panel B-Nil) >8 spots.¹
• The test result is Negative if both (Panel A-Nil) and (Panel B-Nil) <4 spots. This includes values less than zero.¹
• Results where the highest of the Panel A or Panel B spot count is such that the (Panel minus Nil) spot count is 5, 6 or 7 spots should be considered Borderline (equivocal) and 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 TB infection status of the patient.¹

Positive (Abnormal result):¹ Diagnosing or excluding tuberculosis (TB) disease and assessing the probability of latent TB infection (LTBI) requires a combination of epidemiological, historical, medical, and diagnostic findings that should be considered when interpreting T-SPOT^®TB test results. A positive test result does not rule in active TB disease caused by Mycobacterium tuberculosis (Mtb); active TB disease should be confirmed by other tests such as sputum smear and culture, PCR, and chest radiography. Uncommonly, a positive T-SPOT^®TB result may be due to infection with other Mycobacterium species including M kansasii, M szulgai, M gordonae, or M marinum. Alternative tests would be required if these infections are suspected.¹

Negative (Normal result):¹ A negative test result does not exclude the possibility of exposure to or infection with Mtb. Patients with recent exposure to TB-infected individuals exhibiting a negative T-SPOT^®TB result should be considered for retesting within 6 weeks or if other relevant clinical symptoms indicate.¹

Borderline (equivocal result): The patient’s test result cannot be definitively classified as positive or negative. Retesting the patient is recommended although there is no set guideline established for the time interval between an initial borderline result and a retest. If the result is still borderline on retesting with another specimen, then other diagnostic tests and/or epidemiologic information should be used to help determine the Mtb infection status of the patient.¹

Invalid result: The test result is invalid if either the positive or negative control does not meet the quality control acceptance criteria as defined above by the manufacturer. The test result can be invalid because the patient’s lymphocytes either produced gamma-interferon in the absence of antigen or mitogen (Nil/Negative Control failure) OR failed to respond appropriately to mitogen used as a positive control (Positive Control failure). Such invalid results are uncommon. Repeating the test using a new blood sample is recommended around 6 to 8 weeks from the date of invalid test results.¹

As IGRAs are predicated on in vitro release of cytokines (IFN-γ) from stimulated cells, there is likely to be more variability in these tests than those based on the measurement of circulating substances in the blood. The known sources of variability inherent in the IGRAs are^1,2

• The type of measurement itself, i.e., ELISA or ELISPOT
• The reproducibility of a complex biological reaction in a laboratory
• The natural variability of human immune response with viable lymphocytes
• The variability introduced in processing of the blood sample and the test performance.

A T-SPOT^®TB “conversion” refers to a change in a blood test result from negative to positive, indicative of the likely presence of TB infection.² T-SPOT^®TB “reversion” refers to a situation where a blood test result that was previously positive changes to negative after repeating the test. Both ELISPOT conversion and reversion occur after Mtb exposure.² Reversion can be influenced by successful TB treatment, resolution of infections, changes in the patient’s immune system or a false negative result.² On the other hand, conversion can be influenced by the patient’s development of a positive immune response to an active or latent TB infection or a false positive result.²

For more information see T-SPOT^®TB package insert¹ or TB testing guidelines.²

Tuberculin skin test (TST): TST in the United States is the Mantoux method, which involves intracutaneous injection of tuberculin (the purified protein derivative [PPD]) into the volar surface of the forearm. The extent of induration is then read 48-72 hours after administration.³

A prior TST can boost IGRA results.^2,4-7 However, boosting does not occur in uninfected individuals, because they do not have an established immune response to tuberculosis.^2,4-7 Furthermore, the TST, despite its in vivo application, does not cause sensitization or establish a cell-mediated response.^2,4-7 Therefore, it cannot cause subsequent boosting among persons without prior TB infection. 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 nontuberculous mycobacteria or Bacille Calmette-Guérin (BCG) vaccination.^2,4-7

TB vaccine: BCG is a vaccine for tuberculosis that is not generally used in the United States but is routinely administered in over 150 countries with high TB incidence.⁵ The vaccine can cause a false-positive TST reaction.⁵ However, IGRA, which includes T-SPOT^®TB, is the preferred test for individuals who have received the BCG vaccine.^4-7

Live virus vaccination: Vaccination with live viruses, including measles, mumps, rubella, oral polio, varicella, and yellow fever may interfere with TB blood test reactions. For persons scheduled to receive a TB blood test, testing should be done as follows⁵

• On the same day as vaccination with live-virus vaccine, or
• At least one month after the administration of the live-virus vaccine

People at risk for TB fall into two broad categories:⁸

• People who are at higher risk of exposure to TB bacteria
• People who are at higher risk of TB disease developing once infected with TB bacteria.

People at higher risk of exposure to TB bacteria

• Contacts of people known or presumed to have infectious TB disease.
• People who were born in or who frequently travel to countries where TB disease is common
• People who currently live or used to live in large group settings where TB is more common, such as homeless shelters, correctional facilities, or nursing homes.
• Employees of high-risk congregate settings
• Healthcare workers who serve patients with TB disease.
• Populations defined locally as having an increased incidence of latent TB infection or TB disease, possibly including medically underserved populations, low-income populations, or persons with alcohol use or substance use disorders.
• Infants, children, and adolescents exposed to adults who are at increased risk for latent TB infection or TB disease.

People who are at higher risk of developing TB disease once infected with TB bacteria.

• People with HIV
• Children younger than 5 years of age
• People recently infected with TB bacteria (within the last 2 years)
• People with a history of untreated or inadequately treated TB disease
• People who are receiving immunosuppressive therapy such as tumor necrosis factor-alpha (TNF) antagonists, systemic corticosteroids equivalent to/greater than 15 mg of prednisone per day, or immunosuppressive drug therapy following organ transplantation.
• People with silicosis; chronic renal failure; leukemia; or cancer of the head, neck, or lung
• People with diabetes mellitus
• People who have had a gastrectomy or jejunoileal bypass
• People with low body weight (<90% of ideal body weight)
• People who use substances (such as injection drug use)
• Populations defined locally as having an increased incidence of TB disease, including medically underserved and low-income populations.

The T-SPOT^®TB test is not typically indicated for^2.

• People at low risk for TB exposure (CDC discourages routine testing in low-risk individuals) 
• Individuals with documented prior positive TB tests or prior TB treatment