FAQ - Healthcare Professionals
What is the scale of the TB problem?
The World Health Organization (WHO) estimates that more than one-third of the World's population is infected with Mycobacterium tuberculosis. Even in the developed world, TB rates are stable at best, and actually are increasing in many areas. This is due to factors such as immigration, the emergence of drug resistant TB strains, HIV, and other immunosuppressive conditions.
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What are the limitations of the Tuberculin Skin Test (TST)?
- Requires two visits by the patient
- Subjectivity due to inoculation technique and interpretation of results
- May be affected by the BCG vaccine (reducing the specificity of the test and immunosuppressive conditions (reducing the sensitivity of the test)
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Is there a test for latent TB infection?
Until recently, the only way of testing for latent TB was the use of the Tuberculin Skin Test (TST). The introduction of blood based tests (i.e. Interferon Gamma Release Assays or IGRAs), such as the T-SPOT.TB test, provides a new way to diagnose latent TB. The T-SPOT.TB test is an in vitro diagnostic test for the detection of effector T cells that respond to stimulation with MTB specific antigens. These antigens, ESAT-6 and CFP10 are absent from all BCG strains and from most non-tuberculosis mycobacteria. Benefits of the T-SPOT.TB test are:
- Sensitivity of ~ 95%
- Specificity of 99%*
- Reliable in all targeted groups, including†:
- Immunosuppressed
- Immigrants
- Health Care Workers
- TB Suspects
- TB Contacts
- Does not cross react with BCG vaccine and most common nontuberculous mycobacteria
- Single blood test, no repeat visit required.
*T-SPOT.TB Pivotal Clinical Trial. T-SPOT.TB Package Insert; PI-TB-US-V1 when borderline results excluded.
†T-SPOT.TB Pivotal Clinical Trial. Results demonstrated no association with T-SPOT.TB results and immunocompromised status or BCG vaccination. T-SPOT.TB was also not impacted by age. T-SPOT.TB Package Insert; PI-TB-US-V1
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What species of Mycobacteria test positive with T-SPOT.TB?
T-SPOT.TB produces positive results in all three TB-causing species of the Mycobacterium tuberculosis Complex; M. tuberculosis, M. bovis and M. africanum.
The antigens used in the test, ESAT-6 and CFP10, are present in three other atypical or non-tuberculosis mycobacteria; M. marinum, M. szulgai and M. kansasii. The test may produce positive results in patients infected with these three mycobacteria. The antigens used in the test are not present in other non-tuberculosis mycobacteria.
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How many tests can I run with one T-SPOT.TB test kit?
Each 96-well plate will process 24 patient samples. Since no standard curves are required for the T-SPOT.TB test no wells are required for a standard curve so 24 samples can be run with each assay kit however many assay runs are carried out.
The 8-well strip version of T-SPOT.TB (T-SPOT.TB 8) provides the additional flexibility of containing twelve strips of eight wells. Each strip will process 2 samples.
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Can I store blood samples?
Blood samples should be processed within 8 hours.
Addition of the T-Cell Xtend reagent (available from Oxford Immunotec) to the sample immediately prior to processing allows samples to be run in the T-SPOT.TB assay up to 32 hours after they have been collected.
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What blood collection tubes do I use?
Standard blood collection tubes can be used for the T-SPOT.TB test:
- Blood samples can be collected into either sodium citrate or sodium heparin Vacutainer® Cell Preparation Tubes (CPTs) which are available from Becton Dickinson
- Blood samples can be collected into lithium heparin blood collection tubes with PBMCs being subsequently separated using standard separation techniques such as Ficoll-Paque®. Use of heparin tubes also allows samples to be run up to 32 hours after they have been collected through the addition of the T-Cell Xtend reagent to the sample.
Detailed information on sample collection can be found in the T-SPOT.TB Package Insert or Training Guide. See ‘Downloads' for more information.
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CPT and Vacutainer are trademarks of Becton Dickinson
Ficoll-Paque is a trademark of GE Health Technologies
Can I use blood collection tubes that contain EDTA?
EDTA affects the secretion of interferon gamma. Blood collection tubes that contain this anti-coagulant cannot be used.
Detailed information on sample collection can be found in the T-SPOT.TB Training Guide. See 'Downloads' for more information.
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How do I prepare blood samples for the test?
Blood samples are processed using standard laboratory equipement. Detailed information on sample preparation can be found in the T-SPOT.TB Training Guide. See 'Downloads ' for more information.
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Are all the materials required for the test provided in the kit?
The T-SPOT.TB test kit contains the core materials required to successfully run the assay. A list of additional materials needed can be found in the 'Downloads' section.
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Is a standard curve required each time the T-SPOT.TB test is run?
Standard curves are not required since T-SPOT.TB is a test that enumerates T cells. This ensures that 24 samples can be run with each assay kit however many assay runs are performed.
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How do I analyze T-SPOT.TB results?
T-SPOT.TB results are determined by enumerating the spots in the test wells. Spots are counted directly from the well visually, using a magnifying glass, a stereomicroscope, an elispot reader or from a digital image captured from a microscope. Detailed information on counting can be found in the T-SPOT.TB Training Guide, located in the 'Downloads' section.
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Can the finished assay plates be stored for future reading?
Yes, once developed the wells contained in the completed test plates remain stable and do not need to be read immediately. The plates may be archived for retrospective quality control or re-examination for up to 12 months if kept in a dry, dark environment at room temperature.
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What regulatory approvals does the T-SPOT.TB test have?
The T-SPOT.TB test received approval from the US Food and Drug Administration (FDA) on July 30, 2008. The T-SPOT. TB test carries the CE mark which allows it to be marketed in the EU. Approvals have already been achieved in a number of territories outside the EU and approval is being sought in many other countries.
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What is the sensitivity and specificity of the T-SPOT.TB test?
The sensitivity of T-SPOT.TB is 95.6%*
The specificity of T-SPOT.TB is 97.1%*
*T-SPOT.TB Pivotal Clinical Trial. T-SPOT.TB Package Insert; PI-TB-US-V1
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Are there any patients that should be excluded from diagnosis with T-SPOT.TB?
No, T-SPOT.TB can be used in screening of all groups. T-SPOT.TB can be used for HIV-positive persons, recent contacts of TB cases, residents and employees of high risk congregate settings, chronic renal failure, and immunosuppressed patients. In addition, T-SPOT.TB can be used as a diagnostic aid when evaluating patients suspected of having active TB disease.
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Is T-SPOT.TB affected by previous BCG vaccination?
Unlike the TST, there is no association with BCG vaccination and T-SPOT.TB test results. The test utilizes two M. tuberculosis specific antigens (ESAT-6 and CFP10) that do not cross react with the BCG vaccine.
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What is the purpose of cell washing and counting in the T-SPOT.TB test?
Cell washing and counting are very important steps that improve the sensitivity and reliability of the T-SPOT.TB test.
Cell washing removes the background interferon gamma (IFN-γ) that may otherwise produce interference in the test. Removal of this background IFN-γ provides a more reliable "signal". This washing step is absent from ELISA methods.
Cell counting corrects for those samples where the PBMC count is low (e.g. the immunocompromised). After washing, the PBMCs (Peripheral Blood Mononuclear Cells) are counted to ensure that 250,000 cells per well are used in the test.
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What buffer should be used for plate washing with T-SPOT.TB?
Freshly prepared or sterile phosphate buffer saline (PBS) should be used.
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What data is there to support T-SPOT.TB in clinical use?
The body of scientific evidence continues to grow demonstrating the performance of the T-SPOT.TB test. Over 80 peer-reviewed publications showing the test's excellent performance in a wide range of clinical and epidemiological settings have been published:
- Latent TB infection
- TB disease (including pulmonary and extra-pulmonary TB)
- HIV positive and negative patients
- Patients being screened for Anti-TNF-α therapy
- Healthcare worker screening
- Children, adults and the elderly
- Malnourished individuals
- Renal patients undergoing dialysis
- Patients with hematological disorders
- Low prevalence countries in the developed world and high prevalence countries in the developing world
- TB contact tracing
For a summary of the T-SPOT.TB publications click here .
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How simple is T-SPOT.TB to use?
The T-SPOT.TB test is a simplified and validated ELISpot (enzyme-linked immunospot) method, which is similar to an ELISA assay. A whole blood sample is collected from the patient and sent to the laboratory. The laboratory separates the peripheral blood mononuclear cells (PBMCs), washes and counts them. A standard number of these cells are added into microtiter wells where they are exposed to a positive control, nil control and two MTB specific antigens. After overnight incubation a second antibody is added. After incubation the plate is washed and a soluble substrate is added and another incubation occurs. Upon completion of this incubation period the plate is washed and the numbers of spots are enumerated to determine the patient's result.
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Where can I obtain the T-SPOT.TB test?
Outside of North Americal please call our UK office on +44 (0) 1235 442 780 or send an e-mail to info@oxfordimmunotec.com.
In North America, please contact your local Oxford Immunotec Territory Manager at 1-877-20-TSPOT (87768)
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What is the impact of TB testing on healthcare resources?
Studies have shown that the incorporation of T-SPOT.TB in control programs will reduce the overall cost of TB control. This is laregely due to the elimination of significant indirect costs associated with the TST to both health care systems and patients:
- TST requires the person being tested to visit the clinic at least twice. Up to one-third of people do not return to have their test read. This may result in wasted resources and potentially dangerous gaps when containing an outbreak.
- Indirect resource and time costs associated with administering and reading the skin tests are relatively high. These are compounded by the fact that the tuberculin used in the TST, once opened, has to be used within a short time period leading to potential wastage of unused stock.
- False negatives TSTs and non-returners may convert to TB disease leading to morbidity and higher costs of treating disease (including onward transmission).
- False positives TST results, often due to cross reactivity with BCG or environmental mycobacteria can lead to unnecessary anti-TB treatment and associated toxicity testing and clinical follow-up.
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Why is sensitivity important in a diagnostic test for TB?
High sensitivity helps to maximize the reliability of a test for TB, ensuring that the majority of infected people are identified. This is especially important with immunocompromised patients.
A test with poor sensitivity will produce high numbers of false-negative results in people who truly have disease. These subjects will not be treated and so could convert to active TB disease.
In the pivotal clinical study for the T-SPOT.TB test the sensitivity was determined to be 95.6%.
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