The objective of this study was to further investigate the clinical effectiveness of the T-SPOT.TB test in the diagnosis of tuberculosis (TB), including the effects of the T-SPOT.TB test in the evaluation of various types and locations of TB.


We collected 20,332 samples from patients with suspected tuberculosis. Subsequently, we performed an integrative analysis of T-SPOT.TB clinical results and diagnoses, and we evaluated the compositional relationship and positive detection rate of the T-SPOT.TB test in various age groups, sample types, and departments. hospitable. In addition, we compared the number of spots and the composition rate between latent tuberculosis infection (LTBI), active tuberculosis infection, and old tuberculosis infection groups. The active TB group was then divided into pulmonary TB (PTB), pulmonary and extrapulmonary TB (PETB), and extrapulmonary TB (EPTB) subgroups, and we assessed whether there were statistical differences in the number of spots and the composition rate between the subgroups.

  • Subjects

In total, 20,332 samples were collected from patients suspected of having TB. The average age of the participants was 53.15 ± 18.16 years, and they were admitted to the First Affiliated Hospital of Xi’an Jiaotong University from July 2013 to May 2017. Specifically, there were 11,453 men between 6 months and 96 years of age (Average age: 54.70 ± 18.45) and 8,879 women between 3 months and 96 years (Average age: 52.53 ± 17.71 years).

All patients were enrolled according to the following criteria: 1) the subjects did not have heart, liver or kidney disease and did not have an HIV infection; 2) patients were suspected of having TB; 3) The patients were not taking any therapy involving immunosuppression or enhancing medication. Exclusion criteria included: 1) cases lost to follow-up; 2) lost cases due to death; 3) cases that were not diagnosed as TB at the endpoints.

Informed consent was obtained from all participants (informed consent for patients under 16 years of age was obtained from their guardians), and this study was approved by the ethics committee of the First Affiliated Hospital of Xi’an Jiaotong University ( NO. XJTU1AF2018LSK-161).

  • Diagnostic criteria for different groups.

In light of the diagnostic criteria for pulmonary TB outlined by the Ministry of Health of the People’s Republic of China, the included patients were grouped as follows: 1) LTBI: positive tuberculin test and patients with no history of Bacillus Calmette – Guerin ( BCG) vaccination; or the T-SPOT.TB test result was positive and there was no clinical manifestation of TB and the corresponding evidence of aetiology, pathology and imaging. 2) Active TB: This required tubercle bacilli to be detected from bacterial cultures or sputum smears, Langhans caseate or giant cells to be observed by pathological examination, and effective TB therapy with relevant imaging support.

Furthermore, active TB was divided into three subgroups according to disease site: pulmonary TB (PTB), pulmonary and extrapulmonary TB (PETB), and extrapulmonary TB (EPTB). 3) Old TB: this required that the subjects with a history of TB be cured, but pathological alterations were found according to the imaging diagnosis, that no symptoms of TB intoxication were observed and that the aetiology and pathology tests were negative, and that patients with these characteristics were diagnosed with “other diseases”.

  • Collection and handling of samples

Peripheral venous blood samples of 5 ml were obtained from patients with suspected tuberculosis using lithium heparin-anticoagulant tubes. The mononuclear cells were then isolated to prepare a cell suspension. Finally, the T-SPOT.TB assay (Oxford Immunotec, Ltd., Abingdon, UK) was performed as follows: briefly, the cell suspension was seeded on T-SPOT.TB plates and incubated with ESAT-6 ( specific antigen), CFP-10 (specific antigen), positive control or negative control, respectively. Next, a 100 µl cell suspension was added to the corresponding microwells, and these were cultured in a 5% CO2 incubator at 37 ° C.

The microwells were then washed four times with phosphate buffer (PBS). , before adding 50 μl of secondary antibody solution to each well, and the assay was incubated for 1 h at 2-8 ° C. Subsequently, 50 μl of the chromogenic agent was added and the plate was processed avoiding light for 7 to 12 min before finishing with distilled water. The number of spots was measured, where one spot represented a T cell that could secrete specific cytokines. The interpretation of the final results was performed according to the following criteria:

1) The results were considered positive in two scenarios: first if the number of spots in the negative control group was less than 6, and the number of spots in CFP-10 or ESAT-6 wells was 6 points higher than that of the negative control. Second, if the spot count in the negative control group was 6 to 10, and the number of spots of CFP-10 or ESAT-6 was more than two times that of the negative control. 2) Results were considered negative if the number of spots did not meet the above criteria and the positive control functioned normally. Finally, we performed statistical analyzes of the number of spots and the spots represented the proportion of total spots (composition rate) in different types of diseases (LTBI, active TB and old TB) and different subgroups of active TB (PTB, PETB and EPTB).

  • Statistic analysis

All data analyzes were carried out using SPSS software (version 18.0, IBM). A χ2 test was applied to evaluate the comparison analyzes. And the “inspection rate” was calculated (it means the percentage of certain samples among all the samples analyzed). The T-SPOT.TB results showed an asymmetric distribution and were expressed as median and interquartile range. Non-parametric tests were used for comparative analyzes of different groups and a p <0.01 was considered statistically significant.


Positive T-SPOT.TB test results were found in different age groups, sample types, and hospital departments. Elderly patient groups, pleural effusion specimens, and thoracic surgery departments showed the highest rates of positivity. There were no statistically significant differences in the number of CFP-10 and ESAT-6 good spots between disease groups or active TB subgroups. However, the composition rate was significantly different when wells ESAT-6 and CFP-10 were double positive. The number of spots and the rate of composition were statistically different between the three groups of diseases but did not show significant differences between the three subgroups of active TB.


The results of T-SPOT. The TB test showed differences in LTBI, active TB, and old TB. In addition, a higher level of the number of spots was observed in the active TB group.

Leave a Comment