Tuberculosis (TB) remains a major global health concern, necessitating accurate methods for identifying infection. Historically, the Tuberculin Skin Test (TST), also known as the PPD test, was the standard diagnostic tool for detecting exposure to Mycobacterium tuberculosis. Limitations, such as the need for a follow-up visit and susceptibility to false positives, led to the development of modern alternatives. These newer, blood-based diagnostic tools are called Interferon-Gamma Release Assays (IGRAs). The two most widely used IGRAs are the T-Spot.TB test and the QuantiFERON-TB Gold Plus (QFT-Plus) test, and understanding their differences is key for clinical application.
The Shared Science of IGRAs
The foundation of both the T-Spot and QuantiFERON tests is detecting a specific cellular immune response in the patient’s blood. Both assays identify T-lymphocytes (effector T-cells) sensitized by prior exposure to M. tuberculosis. When these memory T-cells encounter specific bacterial proteins in the lab, they reactivate and release chemical messengers. The antigens used are highly specific to the M. tuberculosis complex, primarily targeting Early Secreted Antigenic Target 6 (ESAT-6) and Culture Filtrate Protein 10 (CFP-10).
These proteins are absent from most non-tuberculous mycobacteria and the strains used in the Bacillus Calmette-Guérin (BCG) vaccine. This specificity is an advancement over the TST, which often yielded false positive results in BCG-vaccinated individuals. The common biomarker measured by both tests is interferon-gamma (IFN-g), a cytokine released by the sensitized T-cells. The presence of IFN-g provides an indirect measure of the body’s immune memory, indicating a latent M. tuberculosis infection.
Procedural Differences in Sample Handling
The core distinction between the two assays lies in the way the blood sample is handled and the final measurement technique utilized. The QuantiFERON-TB Gold Plus test (QFT-Plus) employs a whole-blood approach, meaning the patient’s blood is drawn directly into specialized collection tubes containing the M. tuberculosis antigens. A strict requirement for this assay is that the blood must be incubated at 37°C within 16 hours of collection to ensure T-cell viability and responsiveness. After the incubation period, the plasma is separated, and the concentration of interferon-gamma (IFN-g) released is measured using an enzyme-linked immunosorbent assay (ELISA).
The QFT-Plus procedural requirements demand tight logistical control due to the short incubation window, which can pose challenges for remote clinics or during long-distance transport. Conversely, the T-Spot.TB test requires a preliminary step to separate and isolate the Peripheral Blood Mononuclear Cells (PBMCs) from the whole blood sample. This isolation step allows for greater flexibility regarding the time delay between blood collection and assay performance, with some protocols allowing up to 32 hours if PBMCs are properly handled.
The T-Spot assay utilizes an enzyme-linked immunospot (ELISpot) technique. A standardized number of isolated T-cells are plated onto specialized membranes. When the sensitized T-cells release the cytokine, it is captured by an antibody on the membrane, and a subsequent enzyme reaction generates a visible spot. Therefore, while QFT measures the total concentration of IFN-g in the plasma, T-Spot measures the number of individual T-cells actively releasing IFN-g. The final readout is a count of these spots, which correlates to the number of reactive T-cells present in the sample.
Comparing Diagnostic Performance and Interpretation
Both IGRAs demonstrate high and comparable levels of sensitivity and specificity for diagnosing latent tuberculosis infection (LTBI). Their overall clinical performance is similar in healthy, low-risk populations. Differences primarily emerge in specific patient populations regarding the frequency of indeterminate results, which occur when control tubes fail, making the outcome inconclusive and requiring a repeat test.
The QFT-Plus test is more susceptible to indeterminate results in patients with low white blood cell counts, such as those with lymphopenia. This is because QFT relies on a sufficient number of T-cells in the whole blood sample to produce a detectable IFN-g concentration. Conversely, the T-Spot.TB test isolates and standardizes the number of T-cells plated. This standardization generally results in a lower rate of indeterminate results in immunocompromised individuals. For patients with HIV infection or those receiving immunosuppressive therapy, T-Spot often provides a definitive result.
Interpretation also differs structurally. QFT-Plus provides a continuous, quantitative numerical value of the IFN-g concentration. Although interpreted as positive, negative, or indeterminate based on cut-offs, the numerical output can be used in research to monitor immune responses. The T-Spot.TB test yields a qualitative or semi-quantitative result based on the spot count. The spot count is compared to cut-off values, resulting in a binary positive, negative, or borderline determination, which is often simpler for immediate clinical use.
Factors Influencing Test Selection
Test selection depends on logistical constraints and the specific patient population. QFT-Plus is frequently preferred for large-scale screening or settings where immediate processing is guaranteed, due to its simpler whole-blood collection and potential for automation. T-Spot.TB is often chosen when a delay in sample processing is necessary, as isolated PBMCs maintain viability longer than the whole blood required by QFT.
The patient’s immune status is a significant factor. T-Spot is favored for severely immunocompromised patients, such as those with advanced HIV, because of its lower rates of indeterminate results in these groups. QFT-Plus is commonly used when a lower volume of blood is required or when a streamlined, automated workflow is needed for screening large numbers of patients. Other considerations include the local laboratory’s capabilities, staff training, and the relative cost per test.