The T-SPOT.TB test is a unique, single visit blood test for tuberculosis designed to reduce assay variability and maximize sensitivity, even in the immunocompromised.”


TSPOT. TB assay is an IGRA assay used in laboratories for diagnosting Tuberculosis infectious (including latent TB and active TB).


•      Blood collected:

•      Routine phlebotomy

•      Standard collection tube

•      T-Cells:

•      Isolated

•      Washed

•      Counted

•      Standard number T-cells added to specially designed plates.

•      T-Cells stimulated with TB-specific antigens (ESAT-6 and CFP10).

•      T-Cells responding to antigens release interferon-gamma.

•      Interferon-gamma antibodies capture IFN-ꭚ released by T-Cells.

•      Secondary labeled antibody binds to captured IFN-ꭚ.


•      Detection reagent added and reacts with secondary labeled antibody.

•      Reaction produces spots (footprint of where the interferon-gamma was released).

•      Spots are then counted.


Day 1: Cell Isolation

Total elapsed: 2-3 hours

Number of specimens: upto 24 samples

Hands-on time: 2 hours (approx.)

Step 1: Blood Sample Processing

  • Addition of T-Cell Xtend reagent
  • Cell isolation by Ficoll separation
  • Cell washing by centrifugation
  • Cell counting and dilution

Step 2: Plate Set-up

Addition to well:

  • Test specimen
  • TB-specific antigens
  • Positive Control (PHA)
  • Negative Control (AIM V)

After the finsihed the first day proceduce, incubate the plate from 16 to 20 hours.


Total elapsed: 2 hours

Number of specimens: upto 24 samples

Hands-on time: 30 minutes (approx.)

Step 1: Spot Development

  • Plate washing
  • Conjugate & incubate
  • Plate washing
  • Substrate & incubate
  • Plate washing
  • Drying

Step 2: Results

  • Spot Counting
  • Interpretation
  • Reporting

Guideline support TSPOT

Sensitivity data from CE-marked TSPOT.TB Package Insert including immunocompromised groups (CE – pg.10):

Sensitivity was assessed by testing 87 samples from culture confirmed cases of M.tubersulosis infection, including immunocompromised groups. The sensitivity of the T-SPOT.TB test was caculated as 98.8% (86/87) (95% confidence limits 90.8%-99.9%).

IGRA Sensitivity Summary of Head-to-Head Studies in Confirmed TB

The following picture is list of studies of TSPOT.TB’s Sensitivity.

The following is the conclusions from above studies of TSPOT.TB Sensitivity:

  • TB has higher sensitivity
    • Compared to multi-tube ELISA
  • Numerous head-to-head studies
    • Meta-analysis
    • Larger data set = more statistically significant
  • Head-to-head studies in confirmed TB
    • Sensitivity data is better if TB is confirmed
    • TB was confirmed via culture or PCR