What is the intended use of the TSPOT.TB test?

The TSPOT.TB test is an in vitro diagnostic test for the detection of effector T cells that respond to stimulation by Mycobacterium tuberculosis antigens ESAT6 and CFP10 by capturing interferongamma secreted by effector T cells and is intended for use as an aid in the diagnosis of Mycobacterium tuberculosis infection.

The TSPOT.TB test is an indirect test for Mycobacterium tuberculosis infection (including disease) and is intended for use in conjunction with risk assessment, radiography and other medical and diagnostic evaluations.
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What regulatory approvals does the TSPOT.TB test have?

The TSPOT.TB test has carried the CE mark, which allows it to be marketed in the EU, since 2004. Additionally, the TSPOT.TB test received US Food and Drug Administration (FDA) premarket approval in 2008, was approved in China in 2010 and in Japan in 2012. Approvals have also been obtained in many other countries, including, but not limited to, Canada, Taiwan, Russia, Singapore, Thailand, Peru, Nigeria and Mexico.
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What are the advantages of the TSPOT.TB test?

The TSPOT.TB test is the only TB test with sensitivity and specificity exceeding 98%. The TSPOT.TB test is reliable even in challenging testing populations, including BCGvaccinated and immunocompromised persons, and relies on routine phlebotomy procedures.
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How can I start sending samples to the laboratory?

Getting started is a simple, easy process. Contact Oxford Diagnostic Laboratories on 01235 433164 and we will review the steps with you.
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How much does the test cost?

Please contact Oxford Diagnostic Laboratories on 01235 433164. We will put you in touch with your local area representative who will offer you a tailored package, according to your needs.

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When will I get the results?

Assuming overnight shipment, results are usually reported the day following receipt of the sample. If same day samples arrive after test processing start time, they may be carried over until the next day, which means that results will be available one day later than usual. The only exceptions are during public holidays, weekends, the Christmas to New Year period and for highvolume screenings. During these times, we endeavor to return results within 3 working days.
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Do blood samples need to be collected in special collection tubes?

No, Oxford Diagnostic Laboratories accepts samples in standard green top tubes (lithium heparin or sodium heparin). Blood samples may also be collected in sodium citrate tubes.
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Can blood collection tubes containing EDTA (purple top tubes) be used?

No, EDTA (ethylenediaminetetraacetic acid) affects cells’ secretion of interferongamma due to its chelating (calcium binding) properties. Blood collection tubes that contain this anticoagulant therefore cannot be used.
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How much blood is needed for the TSPOT.TB test?

Typically, in immunocompetent patients, sufficient peripheral blood mononuclear cells (PBMCs) to perform the TSPOT.TB test can be obtained with the following agedependent guidelines:

  • Adults and children ≥ 10 years of age: 6 mL
  • Children ≥2 to <10 years of age: 4 mL
  • Children <2 years of age: 2 mL

Please note: The above guidelines may be insufficient in immunocompromised patients with low numbers of PBMCs. Therefore, it may be advisable to collect double the recommended blood volume for immunocompromised patients.
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What information is required on the blood collection tube?

The tube should be labeled with the patient’s identification as well as the date and time of sample collection. This information must correspond to the information on the request form.
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Do you accept other types of specimens beside blood?

No, only whole blood samples collected in lithium heparin, sodium heparin, or sodium citrate tubes are accepted.
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When can samples be sent to Oxford Diagnostic Laboratories?

Samples are accepted before 2:00PM Monday through Saturday (excluding public holidays).
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How should blood samples be stored prior to sending to the laboratory?

Blood samples must be stored at room temperature, between 1825°C, until packaged for transport and should not be centrifuged.

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Which requisition form should be used?

The Oxford Diagnostic Laboratories Request Form should be used.

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After collecting a blood sample, how long do I have to send it to the laboratory?

Blood samples can be shipped on the same day or overnight, as long as they are received by the laboratory no later than 2:00PM the day following venepuncture.
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How are samples shipped to the laboratory?

There are at least 3 shipping methods available to customers. If you need help in choosing a method, or would like help with packing materials, please contact Oxford Diagnostic Laboratories on 01235 433164 for assistance.
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How does DX work?

DX is a courier network usually collecting after 5pm and delivering by 9am next day (may vary depending on location). There is a oneoff fee to register and set up a DX box. Once this is complete, simply place the samples into the box before the scheduled pick up time and a courier will collect the sample(s) and deliver them to Oxford Diagnostic Laboratories. If assistance is needed in setting up a DX account, please contact Oxford Diagnostic Laboratories on 01235 433164.
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How should samples be packaged?

Ensure blood samples are securely packaged in a robust blood transport container for secure and safe transit (in accordance with IATA Packing Instructions 650). Please contact Oxford Diagnostic Laboratories on 01235 433164 for further details.
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How can I obtain dangerous goods training needed to conform to IATA Packaging Instructions 650?

Click here to complete dangerous goods training.
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In which circumstances would the laboratory reject a blood sample?

If a blood sample is rejected, this will be outlined on the report. Additionally, someone may contact you. Examples of reasons why a blood sample would be rejected are listed below:

  • Sample received more than 32 hours after blood collection
  • Sample clotted on arrival
  • Sample frozen on arrival
  • Sample damaged in transit (leakage)
  • Sample received during a public holiday
  • Blood collection tube is not labelled
  • Specimen is collected in tubes that do not contain lithium heparin, sodium heparin or sodium citrate
  • Blood collection tube is underfilled

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How will I receive TSPOT.TB test results?

Test results will be communicated to the designated person(s) identified in the New Customer Registration Form. Results are available via the online results portal, email, post or fax. Please discuss your preferred format with our Customer Support Team on 01235 433164.

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How are TSPOT.TB test results reported?

TSPOT.TB test results are reported as positive, negative, borderline, or indeterminate.
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How are TSPOT.TB test results interpreted?

TSPOT.TB test results are qualitative and are reported as positive, borderline (equivocal) or negative, given that the test controls perform as expected. Test results are determined by firstly enumerating the spots (captured interferongamma from individual T cells) in each of the patient’s four test wells (Positive Control, Nil Control, Panel A, Panel B). Spots can be counted from the test wells using a magnifying glass, stereomicroscope, ELISPOT reader, or a digital image captured from a microscope. Qualitative results are interpreted by subtracting the spot count in the Nil (Negative) Control from the spot count in Panels A and B. Detailed information regarding test result interpretation can be found in the TSPOT.TB package insert.
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What is the sensitivity and the specificity of the TSPOT.TB test?

The sensitivity of the TSPOT.TB test is 98.8% and the specificity of the TSPOT.TB test is 100%.
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Why does the TSPOT.TB test measure interferongamma?

Interferongamma is crucial to immune defense against intracellular pathogens such as Mycobacterium tuberculosis. Post infection, naïve T cells become sensitized to TBspecific antigens and develop into TBspecific effector T cells (both CD4+ and CD8+), which then migrate to the site of infection and secrete interferongamma to activate macrophages to ingest and destroy mycobacteria. TBinfected patients have TBspecific effector T cells circulating in their peripheral blood, which secrete interferongamma in vitro when stimulated by the antigens used in the TSPOT.TB test. The TSPOT.TB test directly captures interferongamma secreted by individual TBspecific effector T cells.
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Why does the TSPOT.TB test have a borderline for interpretation of results?

The vast majority of TSPOT.TB test results are either positive or negative.

A small percentage of test results can be borderline (equivocal), where the higher of (Panel A minus Nil Control) and (Panel B minus Nil Control) is 5, 6 or 7 spots. The borderline category is intended to reduce the likelihood of falsepositive or falsenegative results around the cutoff point of the TSPOT.TB test. As opposed to an indeterminate or invalid result, a borderline result is clinically interpretable and should be followed by retesting as a substantial proportion of individuals may test positive upon retesting. In a study of US healthcare workers, 23% of subjects with a borderline test result retested as positive, suggesting the borderline category is useful in maintaining test sensitivity.1

According to the Centers of Disease Control and Prevention (CDC), “incorporation of a borderline category for the TSPOT[.TB test] as approved by FDA increases test accuracy by classifying results near the cut point (at which small variations might affect the interpretation) as neither positive or negative.”2(8, 12, 19)

1. King TC, Upfal M, Gottlieb A, et al. T-SPOT.TB Interferon-γ Release Assay Performance in Healthcare Worker Screening at Nineteen U.S. Hospitals. Am J Respir Crit Care Med. 2015;192(3):367-373.

2. Mazurek GH, Jereb J, Vernon A, LoBue P, Goldberg S, Castro K, Committee IE, Centers for Disease C, Prevention. Updated guidelines for using Interferon Gamma Release Assays to detect Mycobacterium tuberculosis infection United States, 2010. MMWR Recomm Rep 2010; 59: 125.

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Are borderline TSPOT.TB test results the same as indeterminate results?

No, borderline (equivocal) results are clinically interpretable whereas indeterminate results cannot be interpreted due to the failure of the test’s Positive and/or Nil (Negative) Control. In both cases, however, retesting by collecting another blood sample is recommended.
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What should I do with a borderline TSPOT.TB test result?

Borderline results are clinically interpretable and should be followed. Retesting by collecting another sample is recommended. In a study of US healthcare workers, 23% of subjects with a borderline test result retested as positive, suggesting the borderline category is useful in maintaining test sensitivity1. Upon retesting, if the test result remains borderline, other diagnostic tests and/or epidemiologic information should be used to help determine the TB infection status of the patient.

1. King TC, Upfal M, Gottlieb A, et al. T-SPOT.TB Interferon-γ Release Assay Performance in Healthcare Worker Screening at Nineteen U.S. Hospitals. Am J Respir Crit Care Med. 2015;192(3):367-373.

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What should I do with an indeterminate TSPOT.TB test result?

Indeterminate results are not clinically interpretable and may occur if the Positive and/or Nil (Negative) Control does not perform as expected. Retesting by collecting another sample is recommended. Upon retesting, if the test result remains indeterminate, other diagnostic tests and/or epidemiologic information should be used to help determine the TB infection status of the patient.

Indeterminate results are uncommon and may be related to factors such as inappropriate blood storage conditions, delay in sample transport, patient specific conditions, or laboratory error.

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Why does the TSPOT.TB test include a Positive Control with each patient sample?

The Positive Control serves as an indicator of patient cell functionality. Patient cells are incubated with a nonspecific stimulator, phytohemagglutinin, in the Positive Control sample well. The Positive Control spot count is ≥ 20 in the vast majority of patient samples. A failed Positive Control is uncommon but may be related to factors such as inappropriate blood storage conditions, delay in sample transport, technical factors or patient specific conditions. A small proportion of patients may have T cells which show only a limited response to phytohemagglutinin.

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Why does the TSPOT.TB test include a Nil (Negative) Control with each patient sample?

The Nil (Negative) Control is designed to control for nonspecific T cell reactivity. Patient cells are incubated with sterile media in the Nil Control well. For the test to be considered valid, there must be < 11 spots in the Nil Control well. A failed Nil Control is uncommon but may be related to technical factors or a patient specific condition.
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Are any patient groups excluded from testing with the TSPOT.TB test?

No, the TSPOT.TB test can be used in testing of all patient groups including those living with HIV/AIDS, those with weakened immune systems, recent contacts of TB cases and residents and employees of highrisk congregate settings.
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Can the TSPOT.TB test be used in testing samples from patients with weakened immune systems?

Yes, the TSPOT.TB test is wellsuited for use in patients with weakened immune systems.

Each sample undergoes a cell count which is used to create a normalized (standardized and known number) suspension of cells that are subsequently incubated with TBspecific antigens. Immunocompromised patients may have a reduced number of peripheral blood mononuclear cells (PBMCs) the types of white blood cells used in the TSPOT.TB test. In these patients, multiple blood tubes can be pooled to obtain the required number of cells to perform the test.

Pivotal clinical study data submitted to the US Food and Drug Administration (FDA) for premarket approval extensively evaluated the TSPOT.TB test in immunocompromised individuals including, but not limited to, subjects with HIV, silicosis, diabetes, endstage renal disease and organ transplant. A negative tuberculin skin test was associated with being immunocompromised; while, no association was observed between TSPOT.TB test result and immunocompromised status.
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What is the purpose of the cell washing and counting steps in the TSPOT.TB test?

The cell washing step enables removal of plasma and potentially interfering substances, such as endogenous interferongamma, tricyclic antidepressants, and nonsteroidal antiinflammatory drugs. After washing, the peripheral blood mononuclear cells (PBMCs) are counted to allow for an adjustment in cell concentration to correct for variations in patient PBMC counts and ensure a standard number of cells are used in the test.

The washing and counting steps may be of particular importance in patients with weakened immune systems. Immunosuppressed and immunocompromised patients have a higher risk of progressing from latent TB infection to TB disease, and may be prescribed a potentially interfering substance or have a PBMC count outside of normal values.
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What is the expected frequency of indeterminate results with the TSPOT.TB test?

Indeterminate results are infrequent with the TSPOT.TB test. Oxford Diagnostic Laboratories maintains a low overall indeterminate rate and does not charge for indeterminate TSPOT.TB test results. Contact technical support for additional information.
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Does the TSPOT.TB test differentiate between latent TB infection and TB disease?

No, like a tuberculin skin test (TST) and the ELISAbased TB blood test, the TSPOT.TB test does not differentiate latent TB infection from TB disease.(8, 14)

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How soon after exposure to Mycobacterium tuberculosis can an infection be detected with the TSPOT.TB test?

The time interval for conversion following exposure is not yet well defined, but is expected to occur no later than tuberculin skin test (TST) conversion (typically 28 weeks).
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Is a positive TSPOT.TB test result expected in patients with a previous history of tuberculosis?

Unfortunately, there is no clear answer to this question. Studies do not consistently demonstrate that persons test negative after TB treatment. Clinical cure is described by negative sputum culture, improvement of symptoms and chest xray changes. This term refers to both a sterilizing cure and the return to the quiescent phase (latent TB infection). Some data have shown that a large proportion of individuals remain skin or blood test positive. The persistence of effector T cells, the cells stimulated by TBspecific antigens in the TSPOT.TB test, may suggest the presence of dormant bacteria but further study is required. Depending on requirements, other diagnostic tests and medical examinations could be considered if the patient remains positive after treatment.1-4 

1. Zhang S, Shao L, Mo L, et al. Evaluation of gamma interferon release assays using Mycobacterium tuberculosis antigens for diagnosis of latent and active tuberculosis in Mycobacterium bovis BCGvaccinated populations. Clin Vaccine Immunol. 2010 Dec;17(12):198590.

2. Bosshard V, RouxLombard P, Perneger T, et al. Do results of the TSPOT.TB interferongamma release assay change after treatment of tuberculosis? Respir Med. 2009 Jan;103(1):304.

3. Chee CB, KhinMar KW, Gan SH, et al. Tuberculosis treatment effect on Tcell interferongamma responses to Mycobacterium tuberculosis‑specific antigens. Eur Respir J. 2010 Aug;36(2):35561.

4. Walzl G, Ronacher K, Hanekom W, Scriba TJ, Zumla A. Immunological biomarkers of tuberculosis. Nat Rev Immunol 2011; 11: 343354.

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Can the TSPOT.TB test detect infections of drugresistant tuberculosis strains such as MDRTB, XDRTB or TDRTB?

Yes, the TSPOT.TB test can detect all strains of Mycobacterium tuberculosis complex organisms, including multidrugresistant tuberculosis (MDRTB), extensivelydrugresistant tuberculosis (XDRTB) and totallydrugresistant tuberculosis (TDRTB). The antigens in the TSPOT.TB test are common to all Mycobacterium tuberculosis strains. However, the test does not predict whether the organism is sensitive to usual drug treatments. Drug susceptibility testing occurs after isolation and identification of the organism by other methods.
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Are infections with mycobacteria other than Mycobacterium tuberculosis expected to produce positive TSPOT.TB test results?

Mycobacterium tuberculosis is the causative agent of most cases of tuberculosis and thus, TSPOT.TB test sensitivity was determined from subjects with active, cultureconfirmed Mycobacterium tuberculosis infection. Individuals infected with other Mycobacterium tuberculosis complex organisms (such as M. bovis, M. africanum, M. microti, M. canetti) usually have T cells in their blood which recognize the antigens (ESAT6 and CFP10) used in the TSPOT.TB test and are also anticipated to produce positive TSPOT.TB test results. ESAT6 and CFP10 antigens are absent from most nontuberculous mycobacteria (NTM) with the exception of M. marinum, M. szulgai and M. kansasii. While it is unclear if ESAT6 and CFP10 are present in the genome of all subspecies of M. gordonae, it is possible that this NTM may also produce a positive result. All other nontuberculous mycobacteria, including M. avium, are not expected to crossreact with the antigens used in the TSPOT.TB test.
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Is the TSPOT.TB test affected by previous BCG vaccination?

Unlike a tuberculin skin test, there is no association between BCG vaccination and TSPOT.TB test results.

The bacille CalmetteGuérin (BCG) vaccine is an attenuated derivative of virulent Mycobacterium bovis, the bovine or animal form of the TB mycobacterium. The TSPOT.TB test utilizes antigens (ESAT6 and CFP10) that are located on a genomic region designated as RD1, region of differentiation 1. The RD1 region is present in all virulent M. bovis strains but is deleted from all BCG strains. Because the antigens used in the TSPOT.TB test are not present in the BCG vaccine, the TSPOT.TB test does not produce a falsepositive result due to BCG vaccination. It should be noted, however, that patients infected with virulent M. bovis are likely to produce a positive TSPOT.TB result.
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