Test Principle
	Step 1: Blood Collection
	Step 2: Blood Incubation and Plasma Harvesting
	Step 3. Human IFN- ELISA
	Step 4. Data Analysis

Test Principle

The QuantiFERON-TB Gold In-Tube assay is an in vitro diagnostic laboratory test that aids in the detection of infection with Mycobacterium tuberculosis. It uses human whole blood, with patented assay technology based on the measurement of Interferon-gamma (IFN-) secreted from stimulated T-cells previously exposed to Mycobacterium tuberculosis. The QuantiFERON-TB Gold In-Tube assay is a straightforward laboratory test that involves 4 simple steps:

1. Collection of blood into QuantiFERON-TB Gold In-Tube Blood Collection Tubes.
2. Overnight incubation at 37^oC. TB infected patients' blood cells will produce IFN-.
3. Detection of released IFN- in harvested plasma using a quick and easy ELISA.
4. Analysis of data using the QuantiFERON-TB Gold In-Tube Analysis Software.

Some of the Frequently Asked Questions relating to the assay are listed below. The answers provided act as a guide only. We recommend that the QuantiFERON-TB Gold In-Tube Package Insert be used as the reference for the test procedure, as well as for other enquiries relating to the use or performance of the assay.

Step 1: Blood Collection

The blood hasn't reached the black mark on the side of the Blood Collection Tube. Is this important?
The mark on the side of the tubes indicates the 1 mL fill volume. QuantiFERON-TB Gold In-Tube Blood Collection Tubes have been validated for volumes ranging from 0.8 to 1.2 mL. If the level of blood in any tube is not close to the indicator mark, it is recommended to obtain another blood sample.

How important is the tube mixing process?
The antigen mixing process ensures even distribution of stimulating antigens to allow white blood cells to ingest and process antigen for presentation to T-cells, thus leading to IFN- secretion. It is a very important step in the QuantiFERON- TB Gold In-Tube assay and poor mixing will lead to low and incorrect results.
Mix the tubes by vigorously shaking the tubes 10 times ensuring that the entire surface of the tube has been coated with blood. Thorough mixing is required to ensure complete mixing of the blood with the tube's contents. Causing the blood to froth will not adversely affect the performance of the test. Universal blood handling precautions should be used.
A demonstration video can be viewed on our website at: www.cellestis.com

Can the blood collection tubes be transported lying down?
Yes. QuantiFERON-TB Gold Blood Collection Tubes can be transported lying down only after the tube mixing step has been done. The tubes should be mixed again immediately prior to being placed upright in the 37^oC incubator.

At what temperature can the blood be transported to another site, or held prior to incubation at 37^oC?
Blood should be held and transported at Room Temperature (17^oC to 27^oC). Do not refrigerate the blood or place on ice.

Step 2. Blood Incubation and Plasma Harvesting

What if 37^oC incubation starts more than 16 hours after the time of blood collection?
The Package Insert specifies that the 37^oC blood incubation must commence within 16 hours of collection. Blood samples incubated more than 16 hours after collection are likely to exhibit a decreased IFN- response due to cellular breakdown (death), leading to loss of sensitivity and inaccurate results.

Can I incubate the blood collection tubes lying down?
QuantiFERON-TB Gold Blood Collection Tubes must be kept upright during incubation at 37^oC.

Do I have to centrifuge the tubes before I can harvest the plasma?
While it is recommended to centrifuge the tubes to assist with harvesting, it is possible to harvest the plasma from the tubes without centrifugation. Additional care is required to remove the plasma without disturbing the cells.

Do I have to centrifuge the tubes immediately after removal from incubator?
QuantiFERON-TB Gold Blood Collection Tubes may be held between 2^oC and 27^oC for up to 3 days before centrifugation or harvesting.

The gel plug hasn't moved during centrifugation. What should I do?
After incubation of tubes at 37^oC, the plasma is separated from the cells by centrifuging for 15 minutes at 2000 - 3000 RCF (g). The gel plug should move to separate the cells from the plasma. If this does not occur, the tubes should be re-centrifuged at a higher speed.

The plasma doesn't appear the colour it normally does. Is this OK?
Plasma from the QuantiFERON-TB Gold In-Tube Blood Collection Tubes can appear more red than usual but this is normal. It should be noted that the colour of plasma, even those without any red blood cell contamination, can vary from almost colourless to shades of yellow/pale brown; some plasma samples even have an opaque character. These qualities have not been found to affect QuantiFERON-TB Gold In-Tube results.

Do I require a Class II Biohazard Cabinet in which to perform plasma harvesting?
Ideally, all work with blood should be performed in a Biohazard Cabinet to minimize the risk of infection (eg HIV, Hepatitis-B) from potentially infectious blood samples. However, as long as aseptic techniques are used, plasma harvesting can be performed outside of a Biohazard cabinet. 'Safe Laboratory Practices' should be followed, including the use of protective clothing such as gloves, gown, safety eyewear etc, as suggested by relevant regulators.

When harvesting the plasma from above the sedimented red blood cells or gel plug, what volume do I need to remove? Is this important?
As little as 100 L of plasma is sufficient, as only 50 L of plasma is required to perform the ELISA. 200 L will leave sufficient plasma for reference (re-testing) purposes, if required. It is generally possible to take greater than 300 L. Validation studies have shown that IFN- is evenly distributed in the plasma and the volume removed is not critical. The volume of plasma available can vary from patient to patient.

The QuantiFERON-TB Gold In-Tube method also allows for direct sampling of plasma from the blood collection tube using an automated system. This allows the plasma harvesting and ELISA to be performed with minimal operator input.

I want to maximise the cost effectiveness of the QuantiFERON-TB Gold In-Tube assay by batching my samples. What is the stability associated with the harvested plasma?
Harvested plasma (or plasma stored in the blood collection tubes after centrifugation) can be stored at 2^oC to 8^oC for up to 28 days, or below -20^oC for extended periods; plasma kept at -70^oC are less likely to form clots. For short-term storage (less than 28 days), it is better to refrigerate plasma samples rather than freeze them, due to possible fibrin clot formation.

What should I do if "clots" form in my plasma samples during their storage at -20^oC?
Upon thawing, frozen plasma samples may require centrifugation to sediment the clots that can form during the freeze/thaw process. A guide to dealing with clotted plasma samples is outlined in the Package Insert.

Do I need to use microtubes for storing harvested plasma? Can I use more "cost effective" ELISA plates in this instance?
Uncoated ELISA plates can be used to store harvested plasma. Regardless of type, all plasma storage vessels should be sealed to avoid evaporation of samples.

Step 3. Human IFN- ELISA

	a)	Reconstituted IFN- Standard may be kept for up to 3 months if stored at 2oC to 8oC (the date of reconstitution should be noted). Reconstituted IFN- standard should be equilibrated at Room Temperature (17oC to 27oC) for 1 hour before use.
	b)	Once reconstituted, the Conjugate 100X Concentrate must be used within 3 months or discarded. Working strength conjugate (Conjugate 100X Concentrate mixed with Green Diluent) must be used within 6 hours of its preparation. Any unused Conjugate 100X Concentrate must be returned immediately to 2oC to 8oC following its use.
	c)	Working strength Wash Buffer may be stored at Room Temperature (17oC to 27oC) for up to 2 weeks.

Can I use the QuantiFERON-TB Gold ELISA plates immediately after their removal from the fridge?
No. Sealed ELISA plates should be allowed to equilibrate at Room Temperature for at least 1 hour before opening the foil bag.

Do I require an automated Plate Washer?
No. Although an automated plate washer is recommended, manual washing can be performed following the procedure as outlined in the QuantiFERON-TB Gold In-Tube Package Insert.

How important is washing during the QuantiFERON-TB Gold ELISA?
As with most ELISAs, inadequate or incorrect washing is the single most common cause of QuantiFERON-TB Gold ELISA error. If you have any such problems, please check the following:

• If bubbles and froth form during the wash steps, the flow rate of the wash cycle should be adjusted (usually lowered) to prevent this from occurring.
• Wash volumes should allow the wash buffer to reach the top of each well (preferably with a positive meniscus forming over the rim of each well).
• Ensure all wells receive sufficient and equal wash buffer. Blocked washer probes can be cleaned with a fine wire.
• 6 complete washes are suggested as a minimum in the Package Insert, however additional washes can be performed without affecting the performance of the assay.
• A soak period of at least 5 seconds between each cycle is recommended.
  

My results are not as I had anticipated. What could be the problem?
General ELISA problems with the possible causes and appropriate solutions are listed in the tables below.

Non specific Color Development
POSSIBLE CAUSE	SOLUTION
Incomplete washing of the plate	Wash the plate at least 6 times with 400 L/well of wash buffer.  More than 6 washing cycles may be required depending on the washer being used.  A soak time of at least 5 seconds between cycles should be used.
Cross-contamination of ELISA wells	Take care pipetting and mixing samples to minimize risk.
Components have expired	Ensure kit is used within the expiry date.  Ensure that reconstituted Standard and Conjugate 100X Concentrate are used within three months of the reconstitution date.
Components are contaminated	Avoid contamination of components.

High Background
POSSIBLE CAUSE	SOLUTION
Incomplete washing of the plate	Wash the plate at least 6 times with 400 L/well of wash buffer. More than 6 washing cycles may be required depending on the washer being used.  A soak time of at least 5 seconds between cycles should be used.
Conjugate reconstitution/dilution error	Conjugate 100X Concentrate should be reconstituted with 300 mL of distilled water. Working strength conjugate is prepared by diluting the Conjugate 100X Concentrate 1/100 in Kit Green Diluent as per the Package Insert.
Incubation temperature too high	Incubation of the ELISA should be performed at Room Temperature, 17oC to 27oC.
Components have expired	Ensure that reconstituted Standard and Conjugate 100X Concentrate are used within three months of the reconstitution date.
Enzyme Substrate is contaminated.	Discard substrate if blue coloration exists.  Ensure clean reagent reservoirs are used.

Low Absorbance

Poor Standard Curve

POSSIBLE CAUSE	SOLUTION
Incomplete washing of the plate	Wash the plate at least 6 times with 400 L/well of wash buffer. More than 6 washing cycles may be required depending on the washer being used.  A soak time of at least 5 seconds between cycles should be used.
Standard Dilution Error	Ensure dilutions of the Kit Standard are prepared correctly as per the Package Insert.

Duplicate Variability

Note: An assay procedure video, and solutions to most technical problems, can be found on the Product Information and Technical Guide CD-ROM available from Cellestis.

Step 4. Data Analysis

I have very high Nil control values? What may be the problem?
Under most circumstances, the expected IFN- concentration range for the Nil control is below 8 IU/mL. If Nil control values are much greater than this, the result may be due to a technical error. In such instances judgement should be used in determining whether to re-test, and if required, it is recommended that both of the subject's plasma samples be re-assayed. If the Nil result remains high, and contamination of the sample plasma is unlikely, the Nil result is valid. A good practice is to check all Nil control values following each test to make sure they fall within, or are close to, the expected range.

A patient's TB Antigen value is very high (possibly above the detectable limit of the plate reader). Is this OK?
In some cases the patient's TB Antigen IFN- level may be above the limit of the microplate reader - such an occurrence will have no impact on the test interpretation, provided the result for that patient's Nil Antigen is below 8 IU/mL.

Can the amount of IFN- measured be correlated to the stage or degree of TB infection?
No. Individuals displaying a response greater than or equal to 0.35 IU/mL above the Nil control (and greater than or equal 25% of Nil value), for the TB Antigen, are likely to be infected with M.tuberculosis. No correlation between their response to these antigens and the stage or degree of infection, their level of immune responsiveness, or their likelihood for progression to active disease can be made.

Is there an easy way of calculating and interpreting QuantiFERON-TB Gold In-Tube test results?
An outline of the Data Analysis and Test Interpretation method for the QuantiFERON-TB Gold In-Tube assay is provided in the Package Insert. Calculation of QuantiFERON-TB Gold In-Tube results can be performed using a spreadsheet program.

Alternatively, QuantiFERON-TB Gold In-Tube Analysis Software is available from Cellestis to analyse assay raw data, and calculate patient QuantiFERON-TB Gold In-Tube results.
The QuantiFERON-TB Gold In-Tube Analysis Software allows the simple transfer of raw data (ODs) directly from microplate reader software (or from any spreadsheet program). The Software performs:

• Calculation of a Standard Curve
• Quality Control check of the standard replicates and curve
• Calculation of all sample IFN- concentrations (IU/mL) from the Standard Curve
• Reporting of a diagnostic result for each patient, according to the 'Interpretation of Results' guidelines outlined in the QuantiFERON-TB Gold In-Tube Package Insert.