Latent TB Infection (LTBI)

Useful For

Investigation of tuberculin skin test (TST) positive findings to differentiate latent tuberculosis infection (LTBI) from BCG vaccination.


Indications for Testing

Specific subgroup / clinical indication Recommendation
1 POSITIVE TST in “low risk” adults and children 5 – 17 years old. IGRA1 may be used as a confirmatory test when the only increased risk factor for progression to active disease is cigarette smoking, underweight or abnormal chest x-ray due to granuloma
2 Immunocompromised2 adults and children (<18 years) IGRA may be used in immunocompromised person with a negative initial TST result
3 Healthcare workers, prison inmates and staff, employee screening IGRA may be used as a confirmatory test for a positive baseline TST in an immunocompetent health care worker or prison staff/inmate that is considered to have a low present probability for LTBI and EITHER has no high risk factor, OR is not on treatment with glucocorticoids or TNF-α inhibitors and does not have diabetes mellitus. If an IGRA is negative, this person could be tested again with an IGRA if an exposure occurs (8 weeks post exposure testing)
4 Diagnosis of active TB in children (under age of 18) with suspected TB disease IGRA may be used as a supplementary diagnostic aid in combination with TST and other investigations to help support a diagnosis of TB. A negative IGRA (or TST) does NOT rule out active TB disease.
5 Adult and childhood contacts of a case of active infectious tuberculosis IGRA may be used as a confirmatory test for a positive TST in contacts (adult or child) with a low pretest probability of recently acquired LTBI and with no other high risk factors for progression to active disease if infected.

1 IGRA: Interferon-gamma release assay

2 Immunocompromised refers to HIV infection, organ transplant recipient, immunosupressive drugs.


Special Instructions and Forms

Quantiferon specimens can only be received at the PHL Monday through Thursday (or Monday through Wednesday if Friday is a holiday).

Quantiferon Collection and Shipping Instructions

Preapproval TST Form


Critical Instructions, please read carefully

1.      Tubes must be between 17°C and 25°C at the time of blood filling.

2.      As 1 mL tubes draw blood relatively slowly, keep the tube on the needle for two to three seconds once the tube appears to have completed filling to ensure that the correct volume is drawn (critical for accurate analysis)

3.      The black mark on the side of the tubes indicates the 1mL fill volume. The blood collection tubes are manufactured to draw 1 mL ±10% and perform optimally within the range of 0.8 to 1.2 mL. If the level of blood in any tube is not close to the indicator line, it is recommended that another blood sample be collected using a syringe and 1mL transferred to each of the three tubes. For safety reasons, this is best performed by removing the syringe needle, while ensuring appropriate safety procedures, removing the caps from the three QFT-Gold IT tubes, and adding 1 mL of blood to each (to the black mark on the side of the tube label). Replace the tube caps securely and mix as described in step 2 below. Under- or over-filling the tubes outside of the 0.8 to 1.2 mL range may lead to erroneous results.

4.      If a “butterfly needle” is being used to collect blood, a “purge” tube should be used to ensure that the tubing is filled with blood prior to the QuantiFERON®-TB Gold IT tubes being used.

5.      Immediately after filling tubes, shake them 10 times just firmly enough to ensure that the entire inner surface of the tube is coated with blood to solubilize antigens on tube walls.

6.      Over energetic shaking may cause gel disruption and could lead to aberrant results.

Incubation (by RHA microbiology laboratory)

1.      Place tubes in a 37°C incubator within 16 hours of collection.

*If the blood is not incubated immediately after collection, mix the tubes by gentle inversion ten times prior to incubation.

2.      Incubate the tubes UPRIGHT at 37°C for 16–24 hours. The incubator does not require CO2 or humidification.

Note: If the blood is not incubated immediately after collection, remixing of the tubes by inverting 10 times must be performed immediately prior to incubation.

Post Incubation

1.      After incubating at 37°C, tubes may be held between 2–27°C for up to three days pre-centrifugation.

Note: All tubes MUST be centrifuged before being sent to PHL.

2.      Centrifuge for 15 minutes at 2,000–3,000 RCF (g). If the gel plug does not move to separate the cells from the plasma (as may be evident with refrigerated tubes), the tubes should be recentrifuged at a higher speed. The blood collection tubes contain a gel plug that separates the plasma from the cells when centrifuged.

3.      After centrifugation, the plasma must be stored in original collection tube. Plasma should be stored at 2–8°C before shipping to PHL.

3 ml (3 X 1 mL)


Transport Temperature

Specimen Room temperature Refrigerated Frozen
If sent directly to PHL (received within 16h after blood collection at PHL) YES NO NO
Between collection and incubation at RHA lab (within 16h after blood collection) YES* NO NO
Postincubation; between RHA lab and PHL NO YES NO

*Tubes must be incubated by the RHA laboratory within 16 hours of collection


Method Name

Interferon-gamma release assay (IGRA)

Enzyme linked immunoassay (ELISA)


Reporting Name





Latent tuberculosis infection



Interferon gamma release assay




Specimen Required

Kit contains three tubes:

1.      Nil control (grey cap), requires 1 ml whole blood

2.      TB antigen (red cap), requires 1 ml whole blood

3.      Mitogen control (purple cap), requires 1 ml whole blood

1 ml of whole blood collected by venipuncture directly into each of the three QuantiFERON®-TB Gold IT blood collection tubes (link to supplied entry). Total volume 3 ml.

Specimen Room temperature Refrigerated Frozen
Post incubation, ship to PHL NO YES NO


Reject Due To

Specimens other than QuantiFERON®-TB Gold IT blood collection tubes
Non-incubated specimens Received >16 hours post collection
Non-centrifuged specimens Unacceptable for submission to PHL (post incubation)
Non-incubated specimens Received on Friday or two days preceding a holiday

Useful For

Investigation of tuberculin skin test (TST) positive findings to differentiate latent tuberculosis infection (LTBI) from BCG vaccination.


Clinical Information

When a subject is exposed to M. tuberculosis and infection occurs, the infection may either progress to active tuberculosis (TB) disease, defined as signs, symptoms and/or laboratory abnormalities indicating tissue destruction, or remain in a subclinical asymptomatic state known as latent tuberculosis infection (LTBI). After a period of time in the latent state, individuals with LTBI may progress to active disease. This time period may last from months to many years. TB disease occurring after a period when signs, symptoms and/or laboratory abnormalities indicating tissue destruction are not present and in the absence of re-exposure is the result of reactivation of previously controlled infection and is termed reactivation TB.


The main purpose of diagnosing LTBI is to consider medical treatment for preventing tuberculosis disease. Until recently the tuberculin skin test (TST) was the only available method for diagnosing LTBI. Cutaneous sensitivity to tuberculin develops from 2 to 10 weeks after infection. However, some infected individuals, including those with a wide range of conditions hindering immune functions, but also others without these conditions, do not respond to tuberculin. Conversely, some individuals who are unlikely to have M. tuberculosis infection exhibit sensitivity to tuberculin and have positive TST results after vaccination with bacille Calmette-Guérin (BCG), infection with mycobacteria  other than M. tuberculosis complex, or undetermined other factors.


The QuantiFERON®-TB Gold IT test is a test for Cell Mediated Immune (CMI) responses to peptide antigens that simulate mycobacterial proteins. These proteins, ESAT-6, CFP-10 and TB7.7(p4), are absent from all BCG strains and from most nontuberculosis mycobacteria with the exception of M. kansasii, M. szulgai and M. marinum. Individuals infected with M. tuberculosis complex organisms usually have lymphocytes in their blood that recognize these and other mycobacterial antigens. This recognition process involves the generation and secretion of the cytokine, IFN-γ. The detection and subsequent quantification of IFN-γ forms the basis of this test. The antigens used in QuantiFERON®-TB Gold IT are a peptide cocktail simulating the proteins ESAT-6, CFP-10 and TB7.7(p4). Numerous studies have demonstrated that these peptides antigens stimulate IFN-γ responses in T-cells from individuals infected with M. tuberculosis but generally not from uninfected or BCG vaccinated persons without disease or risk for LTBI. However, medical treatments or conditions that impair immune functionality can potentially reduce IFN-γ responses. Patients with certain other mycobacterial infections might also be responsive to ESAT-6, CFP-10 and TB7.7(p4) as the genes encoding these proteins are present in M. kansasii, M. szulgai and M. marinum.


Reference Values

Quantiferon should be ordered only for TST-positive cases:

(Please visit the Provincial Disease Control Manual for TB control guidelines,


First Dimension of Interpretation of the TST – Size

TST reaction size (mm induration) Situation in which reaction is considered POSITIVE
0 – 4 HIV infection with immune suppression AND the expected likelihood of TB infection is high (e.g. patient is from a population with a high prevalence of TB infection, is a close contact of an active contagious case, or has an abnormal x-ray).
5 – 9 HIV infectionClose contact of active contagious caseChildren suspected of having tuberculosis diseaseAbnormal chest x-ray with fibronodular diseaseOther immune suppression: TNF-α inhibitors, chemotherapy
≥ 10 All others



Diagnosis or excluding tuberculosis disease, and assessing the probability of LTBI, requires a combination of epidemiological, historical, medical and diagnostic findings that should be taken into account when interpreting QuantiFERON®-TB Gold IT results.

POSITIVE:        M. tuberculosis infection likely

NEGATIVE:      M. tuberculosis infection NOT likely

INDETERMINATE: Results are indeterminate for TB antigen responsiveness

·         A negative QuantiFERON®-TB Gold IT result does not preclude the possibility of M. tuberculosis infection or tuberculosis disease: false-negative results can be due to stage of infection (e.g., specimen obtained prior to the development of cellular immune response), co-morbid conditions which affect immune functions, incorrect handling of the blood collection tubes following venipuncture, incorrect performance of the assay, or other immunological variables.

·         A positive QuantiFERON®-TB Gold IT result should not be the sole or definitive basis for determining infection with M. tuberculosis. Incorrect performance of the assay may cause false positive responses.

·         A positive QuantiFERON®-TB Gold IT result should be followed by further medical evaluation and diagnostic evaluation for active tuberculosis disease (e.g., AFB smear and culture, chest x-ray).


Clinical Reference

Cellestis. 2009. QuantiFERON®-TB Gold (In-tube method): package insert. Cellestis Inc. Valencia, CA, USA.


Canadian Tuberculosis Committee (CTC). An advisory Committee Statement: Recommendations on interferon gamma release assyas for the diagnosis of latent tuberculosis infection – 2010 update. Canada Communicable Disease Report. 2010;36.


Menzies D., and Khan, K. Diagnosis of tuberculosis infection and disease. In Long, R., and Ellis, E. Eds. Canadian Tuberculosis Standards 6th Ed. Public Health Agency of Canada, 2007.


Status Days Analytic Time Maximum Laboratory Time Specimen Retention
Routine Wednesday 7h 5 days 1 month


Method Description

The QuantiFERON®-TB Gold IT system uses specialised blood collection tubes, which are used to collect whole blood. Incubation of the blood occurs in the tubes for 16 to 24 hours, after which, plasma is harvested and tested for the presence of IFN-γ produced in

response to the peptide antigens.


The QuantiFERON®-TB Gold IT test is performed in two stages. First, whole blood is collected into each of the QuantiFERON®-TB Gold blood collection tubes, which include a Nil Control tube, TB Antigen tube, and a Mitogen Control tube. The Mitogen tube is used with the QuantiFERON®-TB Gold IT test as a control for correct blood handling and incubation and serves as a control especially where there is doubt as to the individual’s immune status and thus ability to respond in the test. The tubes should be incubated at 37°C as soon as possible, and within 16 hours of collection. Following a 16 to 24 hour incubation period, the tubes are centrifuged, the plasma is removed and the amount of IFN-γ (IU/mL) measured by ELISA.


A test is considered positive for an IFN-γ response to the TB Antigen tube that is significantly above the Nil IFN-γ IU/mL value. The Mitogen-stimulated plasma sample serves as an IFN-γ positive control for each specimen tested. A low response to Mitogen (<0.5 IU/mL) indicates an indeterminate result when a blood sample also has a negative response to the TB antigens. This pattern may occur with insufficient lymphocytes, reduced lymphocyte activity due to improper specimen handling, incorrect filling/mixing of the Mitogen tube, or inability of the patient’s lymphocytes to generate IFN-γ. The Nil sample adjusts for background, heterophile antibody effects, or nonspecific IFN-γ in blood samples. The IFN-γ level of the nil tube is subtracted from the IFN-γ level for the TB Antigen tube and Mitogen tube.


Performing Laboratory Location

Newfoundland & Labrador Public Health Laboratory

St. John’s


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