Interferon-gamma release assays (IGRAs) are tests that have been developed for identifying TB infection (see Chapter 4: Diagnosis of Tuberculosis Infection). They detect cell-mediated immune responses to specific antigens found in MTBC organisms (including virulent M. bovis) but absent from M. bovis BCG, and most non-tuberculosis myobacteria (the antigens are exceptionally present in M. kansasii, M. szulgai and M. marinum). Detection of a response to these antigens indicates present or previous infection with M. tuberculosis. There are two assays currently approved for use in Canada, the QuantiFERON-TB Gold In-Tube Plus assay (QFT-Plus) (Cellestis/Qiagen, Carnegie, Australia) and the T-SPOT.TB (T-SPOT) (Oxford Immunotec, Abingdon, UK).
IGRAs use whole blood samples and may be performed by any licensed laboratory in Canada. They do not require specialized TB and mycobacteriology laboratory expertise or a CL3 laboratory facility. The assays do, however, require standard clinical laboratory expertise in specimen collection, transportation, and performing the assay.
Laboratories should ensure that specimen collection and transportation are standardized. This is because pre-analytical steps, such as tube shaking, delay between blood draw and incubation, and the exact duration of incubation, are known to affect the results.94,95 If portable incubators are used, it is important to make sure that such incubators can accurately stabilize the temperature at 37 °C. Strict quality assurance is necessary to detect unusual patterns in results (such as a spike in the number of indeterminate results due to low mitogen response or high negative control responses), and it is important to run both positive and negative controls with each assay. Specific details of each test can be obtained from the manufacturers’ instructions.
A.4.1. Assay performance, quality assurance and results interpretation: Key technical information
QuantiFERON Gold Plus is the new version of the QuantiFERON Gold TB test. This test has four tubes in total: negative control, TB antigen tube 1 (TB1), TB antigen tube 2 (TB2) and mitogen control. TB1 contains peptides from ESAT-6 and CFP-10, which are designed to elicit an immune response from CD4+ T-helper lymphocytes. TB2 contains an additional set of proprietary peptides to ESAT-6, CFP-10, which together elicit interferon-gamma (IFN-γ) secretion from both CD4+ and CD8+ T lymphocytes.
Blood may be collected directly into the 4 QFT-Plus test tubes or into a single lithium heparin tube, and then transferred to QFT-Plus tubes. Blood specimens collected in lithium-heparin tubes may be stored at 2-8 °C for up to 48 hours prior to transfer to QFT-Plus tubes.
Result reporting and interpretation for QFT-GIT: Refer to manufacturer’s package insert (reproduced as Table 3). The results of the QFT-Plus assay are defined as positive if either or both of the TB antigen tubes (TB1 and/or TB2) are positive.
While the QFT assay positive cutoff is IFN-γ 0.35 IU/mL for either TB1-Nil or TB2-Nil, it is important to provide to clinicians who have requested this test the actual numerical value of the result (quantitative value), as well as the interpretation (positive, negative, indeterminate). It is recommended that IFN-γ values of 0.20-1.00 IU/mL for QFT be interpreted cautiously. High rates of IGRA conversions, reversions and imperfect reproducibility have been reported in the literature with results in this range. Yet results in this range have also been found to be clinically significant in specific higher risk populations. Hence the positive predictive value of results in this range will vary according to clinical risk factors (see Chapter 4: Diagnosis of Tuberculosis Infection).
Guidance should be provided for an indeterminate result as per the manufacturer’s instructions:
high Nil (high background interferon production) — does not allow an interpretation to be made
low Mitogen (lack of response to antigen stimulation) — does not allow an interpretation to be made
Unreliable or indeterminate results may be due to:
technical failure, including improper protocol
excessive levels of circulating IFN-γ or the presence of heterophile antibodies
greater than 16 hours between time of blood draw and incubation at 37 °C
storage of blood outside ambient temperature range (17 -25 °C)
insufficient mixing of blood collection tubes
incomplete washing of the ELISA plate.
If an indeterminate result is suspected as a result of technical protocol issues (eg, plate washing), repeat testing.
The T-SPOT96–98 assay uses the ELISPOT technique, which involves incubating a defined number of peripheral blood mononuclear cells (PBMCs) with CFP-10 and ESAT-6 antigens. The T cells that have previously been sensitized to M. tuberculosis antigens as a result of infection secrete IFN-γ in response to in vitro stimulation with CFP-10 and ESAT-6.
T-SPOT requires standard blood specimen collection into lithium heparin tubes. After collection, PBMC are separated and enumerated.
T-SPOT requires an inoculum of 1 × 106 viable PBMCs per patient (2.5 × 105 per well, for a total of four wells (nil, ESAT-6 antigen, CFP-10 antigen) and positive control (phytohaemagglutinin, or PHA). PBMCs added to the wells are affixed to a well membrane. Cells responding to the antigens release IFN-γ in the vicinity of their location on the membrane, which is subsequently visualized using anti-IFN-γ antibodies. Results are quantified by counting spots produced where IFN-γ is released. For T-Spot result interpretation, see Figure 1.
Typical results have few or no spots in the nil control.
A nil control spot count in excess of 10 spots should be considered as “indeterminate.”
If a high numbers of spots or a dark background is observed in the nil control wells, the assay reagents and culture media should be checked for contamination.
Greater than 20 spots should be counted in the positive control.
When the positive control is less than 20 spots, it is considered “indeterminate” (unless panel A or B are “reactive” as per the result reporting, see Figure 1); check to ensure that recommended incubation conditions were used. Weak PHA responsiveness may reflect anergy in the patient.
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