QuantiFERON TB Gold In Tube Method (QGIT Test) Manufacturer: Cellestis Inc. (USA) a QIAGEN Company

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1 QuantiFERON TB Gold In Tube Method (QGIT Test) Manufacturer: Cellestis Inc. (USA) a QIAGEN Company P1078 TB APPRISE TB Ante vs. Postpartum Prevention with INH in HIV Seropositive mothers and their Exposed infants

2 Principle and Intended Use QuantiFERON TB Gold In Tube (IT) is an in vitro diagnostic test using a peptide cocktail simulating ESAT 6, CFP 10 and TB7.7(p4) proteins to stimulate cells in heparinized whole blood. Detection of interferon gamma (IFN γ) by Enzyme Linked Immunosorbent Assay (ELISA) is used to identify in vitro responses to these peptide antigens that are associated with Mycobacterium tuberculosis infection. QuantiFERON TB Gold IT is an indirect test for M. tuberculosis infection (including disease) and is intended for use in conjunction with risk assessment, radiography and other medical and diagnostic evaluations.

3 Explanation of the test The QuantiFERON TB Gold IT test is a test for Cell Mediated Immune (CMI) responses to peptide antigens that simulate mycobacterial proteins. These proteins, ESAT 6, CFP 10 and TB7.7(p4), are absent from all BCG strains and from most non tuberculous mycobacteria with some exceptional strains. Individuals infected with M. tuberculosis complex organisms (M. tuberculosis, M. bovis, M. africanum, M. microti, M. canetti) usually have lymphocytes in their blood that recognize these and other mycobacterial antigens. This recognition process involves the generation and secretion of the cytokine, IFN gamma. The detection and subsequent quantification of IFN gamma forms the basis of this test.

4 Blood Collection Incubate at Collection site Ship incubated tubes to testing laboratory Incubate at Laboratory

5 QFT Blood Collection & Tube Handling

6 Blood Collection Tubes Store blood collection tubes at 4 C to 25 C. TB Gold IT blood collection tubes are suitable for use at altitudes between sea level and 2,650 feet. To ensure that the correct volume of blood is drawn between altitudes of 3,350 and 6,150 High Altitude (HA) tubes should be used. The HA tubes are distinguished from Normal tubes by the yellow ring in the cap. At altitudes between 2,650 and 3,350 feet or above 6,150 feet, it is recommended to collect blood using a syringe as described in Section 5 to ensure that the correct volume of blood is added to the tubes.

7 Blood Collection Tubes Filled Volume The black mark on the side of the tubes indicates the 1mL fill volume. The blood collection tubes are manufactured to draw 1mL ± 10% and perform optimally within the range of 0.8 to 1.2mL. If the level of blood in any tube is not close to the indicator line, it is recommended to obtain another blood sample or to collect blood via a syringe as described in the insert. Under or over filling of the tubes outside of the 0.8 to 1.2mL range may lead to erroneous results.

8 A correctly mixed tube Blood Collection Tubes Mixing of Tubes Antigens have been dried onto the inner wall of the blood collection tubes. So it is essential that the tubes contents be thoroughly mixed with the blood. Thorough mixing will dissolve the heparin, preventing clotting, and allow re solubilization of the stimulating antigen. Mixing is performed by shaking the tubes ten (10) times just firmly enough to ensure that the entire inner surface of the tube is coated with blood. Over energetic shaking may cause gel disruption and could lead to aberrant results.

9 Blood Collection Tubes Incubation The tubes must be transferred to a 37 C +/ 1 C incubator as soon as possible, and within 16 hours of collection. Prior to incubation, maintain tubes at room temperature 22 C +/ 5 C) Do not refrigerate or freeze the blood samples. Tubes should be incubated upright. If the blood is not incubated immediately after collection, mixing of the tubes by inverting 10 times must be performed immediately prior to incubation.

10 Option 1: Incubate at Collection Site Blood Collection WARNING: Standard blood handling precautions apply. Collect 1mL blood by venipuncture into each QFT blood collection tube. Tubes should be at 17 to 25 C at the time of blood filling. Tubes fill slowly hold tube on needle for 2 to 3 seconds after flow ceases. If blood level is not close to the black mark on the side of the tube label, obtain another sample. Technical Tip: Butterfly needles prime tubing with a purge tube (not supplied) before filling QFT tubes.

11 Option 1: Incubate at Collection Site Immediately after filling, shake tubes ten times (10x) just firmly enough to ensure that the inner surface of the tube is coated in blood (to dissolve antigens on tube walls). Over energetic shaking may cause gel disruption and could lead to aberrant results. Label tubes appropriately. Blood must be incubated as soon as possible (and within 16 hours of collection). Incubate tubes upright at 37 C for 16 to 24 hours. Humidity/CO2 not required. Note: Portable incubators are available from Cellestis.

12 Option 1: Incubate at Collection Site If tubes are not incubated at 37 C soon after collection, re mix tubes by inverting ten times (10x) immediately prior to incubation. Ship incubated tubes to testing laboratory. Ship within 3 days, if not centrifuged. Maintain tubes at 4 to 27 C. Technical Tip: Label tubes as Incubated.

13 Option 2: Incubate at Laboratory Blood Collection WARNING: Standard blood handling precautions apply. Follow the blood collection step as outline in Option 1. Immediately after filling, shake tubes ten times (10x) just firmly enough to ensure that the inner surface of the tube is coated in blood (to dissolve antigens on tube walls). Over energetic shaking may cause gel disruption and could lead to aberrant results. Label tubes appropriately.

14 Option 2: Incubate at Laboratory Ship tubes to laboratory at 17 to 27 C. Blood must be incubated at 37 C as soon as possible (and within 16 hours of collection). Re mix tubes by inverting 10 times immediately prior to incubation. Technical Tip: Label tubes as Not Incubated.

15 Quick Assay Guide Option 1: Receive Incubated Tubes Centrifuge: Centrifuge tubes at 2000 to 3000g (RCF) for 15 minutes. Centrifuged tubes should be handled with care to avoid mixing of plasma. Plasma in centrifuged tubes is stable for up to 28 days when stored at 2 to 8 C. Harvest Plasma: After centrifugation and prior to harvesting, avoid pipetting up and down or mixing plasma by any means. At all times, take care not to disturb material on the surface of the gel. Plasma samples should only be harvested using a pipette.

16 Quick Assay Guide Option 1: Receive Incubated Tubes Plasma samples can be loaded directly from centrifuged blood collection tubes into the QFT ELISA plate, including when automated ELISA workstations are used. Plasma samples can be stored for up to 28 days at 2 to 8 C or, if harvested, below 20 C for extended periods.

17 Quick Assay Guide Option 1: Receive Incubated Tubes Perform ELISA (a) Add 50μL of working strength conjugate to each well of the QFT ELISA plate. (b) Add 50μL of test plasma and standards to appropriate wells. Incubate for 120 minutes at room temperature. (c) Wash plate 6 times. Add 100μL of substrate. Incubate 30 minutes at room temperature. (d) Add 50μL of stop solution. Read absorbance at 450nm (620 to 650nm ref). Calculate Results: Calculate results using the most current version of the QuantiFERON TB Gold Analysis Software for your region.

18 Quick Assay Guide Option 2: Receive Unincubated Tubes Invert, Incubate, Centrifuge (a) Invert tubes 10 times. (b) Incubate tubes upright at 37 C for 16 to 24 hours. (c) Centrifuge tubes at 2000 to 3000g (RCF) for 15 minutes. Centrifuged tubes should be handled with care to avoid mixing of plasma. Plasma in centrifuged tubes is stable for up to 28 days when stored at 2 to 8 C.

19 Quick Assay Guide Option 2: Receive Unincubated Tubes Harvest Plasma: After centrifugation and prior to harvesting, avoid pipetting up and down or mixing plasma by any means. Plasma samples should only be harvested using a pipette. At all times, take care not to disturb material on the surface of the gel. Plasma samples can be loaded directly from centrifuged blood collection tubes into the QFT ELISA plate, including when automated ELISA workstations are used. Plasma samples can be stored for up to 28 days at 2 to 8 C or, if harvested, below 20 C for extended periods.

20 Quick Assay Guide Option 2: Receive Unincubated Tubes Perform ELISA (a) Add 50μL of working strength conjugate to each well of the QFT ELISA plate. (b) Add 50μL of test plasma and standards to appropriate wells. Incubate for 120 minutes at room temperature. (c) Wash plate 6 times. Add 100μL of substrate. Incubate 30 minutes at room temperature. (d) Add 50μL of stop solution. Read absorbance at 450nm (620 to 650nm ref). Calculate Results: Calculate results using the most current version of the QuantiFERON TB Gold Analysis Software.

21 Unreliable or Indeterminate Results May occur due to Deviation to the procedure as per package insert Incorrect transport/handling of the blood specimen Excessive level of circulating IFN gamma or presence of heterophile antibodies Longer than 16 hours of blood specimen drawing to incubation at 37+/ 1 C All the reagents should be within expiry and should be used as mentioned in package insert and manufacturer s recommendations.

22 References For comprehensive instructions for use, please refer to the Package Insert, available at: Detailed QuantiFERON TB Gold (QFT) information and resources are available in the manufacturer s website (Cellestis) at: A reference library for QFT related information is also available through: (Go to to sign up for your personal Gnowee access.) QFT instructional videos are available at the following links: Blood draw and incubation oiq&index=1&feature=plcp oiq&index=2&feature=plcp Reagents and reconstituting oiq&index=3&feature=plcp See the P1078 MOP for a more detailed list of QGIT references.

23 Special P1078 specific Guidelines QGIT testing should be done in real time. Follow the procedure for Incubation of Blood and Harvesting of Plasma in the QGIT package insert. If unable to run real time or batch testing will be done, follow the guidelines below. Follow the procedure for Incubation of Blood and Harvesting of Plasma in the QGIT package insert. Harvested plasma (or plasma stored in the blood collection tubes after centrifugation) can be stored at 2 C to 8 C for up to 28 days or below 20 C for extended periods. Refer to the QGIT manufacturer s instructions for the details for batch testing within 90 days of collection.

24 Special P1078 specific Guidelines Reporting QGIT Results and Maintaining Blinding of Clinical Site Staff The clinical site must remain blinded to the QGIT result. DO NOT report results of QGIT testing to the clinical site staff. Submit all completed CRFs and results to or fax to (716) This portion to be completed by the site clinical staff. CRF TBW0060 Commercial Interferon Gamma Release Assay (IGRA) Tracking and Test Results

25 Special P1078 specific Guidelines Reporting QGIT Results and Maintaining Blinding of Clinical Site Staff This portion to be completed by the site laboratory staff. CRF TBW0060

26 QGIT Supernatant Storage Following incubation, spin the tubes in centrifuge at 2000 to 3000 RCF for 15 minutes and collect supernatant. Refer QGIT package insert instructions. Store supernatant from the TB antigen tube and negative control only. Divide supernatant equally into 2 aliquots with care not to disturb the cell layer. The site can consider preparing 2x100µl for QGIT testing and remainder supernatant in one cryovial (200 to 300µl) for storage.

27 QGIT Supernatant Storage Store first aliquot of supernatant until testing as per manufacturer s instruction. Store remaining aliquot at 70 0 C or colder until shipping. LDMS spec. code : BLD/QFT/PL1/NIL (from Grey cap tube) BLD/QFT/PL1/ATG (from Red cap tube)

28 QGIT Supernatant Storage If MTA is not required OR already executed, please ship directly to the testing laboratory specified below (and in Section 5 of the LPC): Batch ship remaining plasma aliquot to LDMS Lab# 536 SUN Immunology Research Group (SUN IRG) TYGERBERG 7505, South Africa. Contact: Belinda Kriel Back up contact: Dr Andre Loxton Complete Master Specimen Tracking CRF F3008. Processing labs to complete the remainder of TBW0060 and send to FSTRF.

29 QGIT Supernatant Storage If no MTA executed with the testing Labs, ship ALL samples to BRI as Pass Through shipments. Refer to instructions in the LPC under Section 3 and shipping Instructions under Section 5. BRI, LDMS# 999 John C. Ward Biomedical Research Institute (BRI), USA Complete Master Specimen Tracking CRF F3008. Processing labs to complete the remainder of TBW0060 and send to FSTRF.

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