What happens when a test for tuberculosis infection can't give you an answer?
For clinicians and laboratories screening for TB infection, an indeterminate or invalid result isn't a neutral outcome. It means a repeat blood draw, a delayed decision, and a patient waiting for the next steps. To make things worse, indeterminates and invalids can occur more frequently in the patients who need an accurate and timely diagnosis the most: the immunosuppressed, children, and other patients at the highest risk of progressing to active disease.
With this in mind, evaluating an interferon-gamma release assay (IGRA) goes beyond accuracy alone. Equally important is its ability to provide an actionable result, ensuring clinicians can make informed and timely decisions with confidence. Across a growing body of clinical evidence, the T-SPOT.TB test shows a consistent pattern – low indeterminate rates, even in the more challenging to diagnose groups. In this blog we discuss the evidence.
What causes an indeterminate result?
Every IGRA depends on the same thing: viable T cells, that are healthy enough to respond to stimulus by producing interferon-gamma. The basis of IGRA technology is that you expose T cells to TB-specific antigens, on the assumption that any cell that has encountered TB in the past will respond by producing interferon-gamma. IGRAs measure the interferon-gamma that these cells release, and this provides an indirect measure of whether someone has been infected with TB. But when there are not enough cells, or the cells are not functioning properly, this may lead to an indeterminate test result. Specifically, an indeterminate result occurs either when not enough cells respond in the positive control, or too many cells respond in the negative control. A positive control failure can be the result of too few cells, or weak cell functioning, whilst a negative control failure indicates a non-specific interferon-gamma response. Regardless of the cause, indeterminate results are a challenge for the labs and clinicians performing testing for TB infection, and as the studies discussed in this blog show, the rate varies widely depending on the test used and the patient being tested.
This is where test design starts to matter.
The T-SPOT.TB test works differently from whole-blood IGRAs. Before the cell stimulation step is performed, peripheral blood mononuclear cells are separated from the whole blood sample, washed, and counted. This enables a standardized number of cells to be used in every test well, regardless of how immunosuppressed or lymphopenic the patient is. The assay is then run on this standardized number of cells. Conversely, whole-blood IGRAs test a specific volume of blood, rather than a specific cell number. This subtle difference makes the T-SPOT.TB test less vulnerable to the low cell counts that drive indeterminate results in the patients hardest to test.
By controlling the number of cells tested, the T-SPOT.TB test removes a key cause of indeterminate results. It's also why the patterns demonstrated in the studies discussed in this blog aren't coincidence. Low indeterminate rates, resilience in challenging patients, the ability to resolve a result when a different IGRA test has delivered an indeterminate result1: they all trace back to this methodology that is designed to maintain performance even in the most difficult to diagnose.
Demonstrating reliability at scale
One of the largest studies of T-SPOT.TB reliability comes from a single high-volume reference laboratory. A study by Rego and colleagues gathered 645,947 T-SPOT.TB results. In one of the largest IGRA datasets ever published, the indeterminate rate for T-SPOT.TB came in at 0.6%2. This suggests that the T-SPOT.TB test is a reliable tool for testing in the general population, and for testing at scale, which is required for TB screening programs and outbreak control.
| T-SPOT.TB result | Share of 645,947 tests |
|---|---|
| Negative | 93.5% |
| Positive | 4.0% |
| Borderline | 1.8% |
| Invalid (Indeterminate) | 0.6% |
A robust test for pediatric populations
Young children are one of the groups where guidelines have been most cautious about testing with IGRAs. The concern is that immature immune systems may produce unreliable results, and an indeterminate result in a child means another visit, another blood draw, and ultimately a delay in diagnosing a patient who may be at real risk. As such, a low indeterminate rate is especially important here.
Mandalakas and colleagues analyzed roughly 44,000 T-SPOT.TB results from children in the USA, including more than 5,000 from children under five and over 1,400 under the age of two3. The test returned an actionable result in 98% of cases. The indeterminate (described as invalid in the publication) rate was below 1% for children older than a year, and 1.8% for those under one: a low figure even at the youngest, most challenging end of the range.
| Total results | Positive n (%) | Negative n (%) | Borderline n (%) | Invalid n (%) | |
|---|---|---|---|---|---|
| < 1 year | 455 (1) | 11 (2.4) | 433 (95.2) | 3 (0.7) | 8 (1.8) |
| > 1 < 2 years | 964 (2.2) | 13 (1.3) | 937 (97.2) | 7 (0.7) | 7 (0.7) |
| > 2 < 3 years | 1,047 (2.4) | 31 (3.0) | 1,000 (95.5) | 7 (0.7) | 9 (0.9) |
| > 3 < 5 years | 2,591 (5.9) | 106 (4.1) | 2,453 (94.7) | 19 (0.7) | 13 (0.5) |
| > 5 < 17 years | 27,894 (63.8) | 1,565 (5.6) | 25,829 (92.6) | 353 (1.3) | 147 (0.5) |
| > 10 < 17 years | 27,894 (63.8) | 1,,565 (5.6) | 25,829 (92.6) | 353 (1.3) | 147 (0.5) |
| Total | 43,697 (100) | 2,189 (5.0) | 40,753 (93.3) | 501 (1.1) | 254 (0.6) |
The hardest test: immunosuppressed patients
Immunosuppressed patients are another cohort that are challenging to diagnose with IGRAs, and are at high risk of progressing from TB infection to disease. Low lymphocyte counts, which can occur in those suffering from immunosuppression, can cause difficulty when using immune-based tests and can be a factor in causing indeterminate IGRA results.
Batista and colleagues reviewed 1,599 IGRA tests in 1,299 cancer patients at a major US cancer center, patients were tested with either a T-SPOT.TB test or a whole blood IGRA, QuantiFERON-TB (QFT)4. Across 1,013 T-SPOT.TB tests, 89% returned an actionable result, positive or negative. Across 586 QFT test results, 65% were actionable.
| T-SPOT.TB (n=1,013) | QuantiFERON-TB (n=586) | |
|---|---|---|
| Actionable result (positive or negative) | 89% | 65% |
| Indeterminate result | 10% | 35% |
For QFT, more than a third of tests (35%) returned an indeterminate result, compared with 10% indeterminate results for the T-SPOT.TB test. Markers of a weakened immune system (low albumin, low hemoglobin, low lymphocyte counts) were linked to indeterminate results on both tests. These immune factors affected the T-SPOT.TB test less, consistent with a design that standardizes the number of cells tested rather than relying on the patient's own cell count.
Breaking the diagnostic deadlock
A further study demonstrated that in patients who had received an indeterminate result using a whole-blood IGRA, the T-SPOT.TB test was often able to resolve the result to provide a clear positive or negative. At a hospital laboratory in Bergamo, Italy, QuantiFERON-TB Plus was the routine first-line IGRA. Over seven years, it produced an indeterminate result for 576 patients. Pagnoncelli and colleagues investigated what happened when those patients were retested with the T-SPOT.TB test1.
The T-SPOT.TB test resolved 87.6% of them: 120 of 137 previously indeterminate cases received a clear positive or negative with T-SPOT.TB. When the T-SPOT.TB test was run within 30 days of the original QFT-Plus, it resolved 84.8% of the indeterminate results. In short, when these indeterminate cases were retested with the T-SPOT.TB, it returned a definitive result in the large majority of them.
On the back of this result, the authors proposed adding the T-SPOT.TB test to the diagnostic algorithm specifically for patients whose QFT-Plus comes back indeterminate.
Fewer dead ends, clearer decisions
Across four independent studies covering routine screening, children, immunosuppressed patients, and patients who had previously received indeterminate results, the T-SPOT.TB test shows the same pattern: it returns a result clinicians can act on in the large majority of cases.
That consistency reflects how the test is designed. By preparing a standardized number of cells for every test, the T-SPOT.TB test is less dependent on the patient's own immune status than whole-blood assays, allowing it to return an actionable result across a wide range of patient populations.
The largest of these studies puts a figure on it. Across 645,947 consecutive tests in a single reference laboratory, Rego and colleagues reported a T-SPOT.TB test indeterminate rate of 0.6%2. Below 1%, in one of the largest IGRA datasets published.
For the labs and clinicians performing TB infection testing, and for the patients being tested, that reliability is important.
Want to see what consistent, actionable results could mean for your lab? Get in touch with the Revvity team to learn more about the T-SPOT.TB test.
References:
- Pagnoncelli, M., Arosio, M., Genovesi, A., Napolitano, G. & Farina, C. Performance of the T-SPOT.TB test in patients with indeterminate QuantiFERON-TB Gold Plus results: proposal for an algorithm for the diagnosis of Latent Tuberculosis Infection. Infez. Med. 32, 525–531 (2024).
- Rego, K., Pereira, K., MacDougall, J. & Cruikshank, W. Utility of the T-SPOT®.TB test’s borderline category to increase test resolution for results around the cut-off point. Tuberculosis 108, 178–185 (2018).
- Mandalakas, A. M., Highsmith, H. Y., Harris, N. M., Pawlicka, A. & Kirchner, H. L. T-SPOT. TB Performance in Routine Pediatric Practice in a Low TB Burden Setting. Pediatric Infectious Disease Journal 37, 292–297 (2018).
- Batista, M. V. et al. The Utility of Interferon-γ Release Assays in the Diagnosis of Tuberculosis in Patients With Cancer. Transplant Infectious Disease 27, e14428 (2025).
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Revvity Inc. does not endorse or make recommendations with respect to research, medication, or treatments. All information presented is for informational purposes only and is not intended as medical advice. For country specific recommendations, please consult your local health care professionals.
