QuantiFERON–CMV is not available in the United States.
QuantiFERON–CMV is CE marked for commercial use in Europe


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Test Principle 2013 The QuantiFERON-CMV assay is an in vitro research test that detects CD8+ T cell immunity to human cytomegalovirus infection (HCMV). It uses human whole blood, with patented assay technology based on the measurement of Interferon-gamma (IFN-ɣ) secreted from stimulated T-cells previously exposed to HCMV. The QuantiFERON-CMV assay is a straightforward laboratory test that involves 4 simple steps: 1. Collection of blood into QuantiFERON-CMV Blood Collection Tubes. 2. Overnight incubation at 37°C. HMCV infected patients' blood cells will produce IFN-ɣ 3. Detection of released IFN-ɣ in harvested plasma using a quick and easy ELISA. 4. Analysis of data using the QuantiFERON-CMV 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-CMV 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.
The blood hasn't reached the black mark on the side of the Blood Collection Tube. Is this important? 2013 The mark on the side of the tubes indicates the 1 mL fill volume. QuantiFERON-CMV 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? 2013 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-ɣ secretion. It is a very important step in the QuantiFERON- CMV 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.
Can the blood collection tubes be transported lying down? 2013 Yes. QuantiFERON-HCMV 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°C incubator.
At what temperature can the blood be transported to another site, or held prior to incubation at 37°C? 2013 Blood should be held and transported at Room Temperature (17°C to 27°C). Do not refrigerate the blood or place on ice.
What if 37°C incubation starts more than 16 hours after the time of blood collection? 2013 The Package Insert specifies that the 37°C 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-ɣ response due to cellular breakdown (death), leading to loss of sensitivity and inaccurate results.
Can I incubate the blood collection tubes lying down? 2013 QuantiFERON-HCMV Blood Collection Tubes must be kept upright during incubation at 37°C.
Do I have to centrifuge the tubes before I can harvest the plasma? 2013 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? 2013 QuantiFERON-HMCV Blood Collection Tubes may be held between 2°C and 27°C for up to 3 days before centrifugation or harvesting.
The gel plug hasn't moved during centrifugation. What should I do? 2013 After incubation of tubes at 37°C, 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? 2013 Plasma from the QuantiFERON-CMV 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-CMV results.
Do I require a Class II Biohazard Cabinet in which to perform plasma harvesting? 2013 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? 2013 As little as 100 µLof plasma is sufficient, as only 50 µL of plasma is required to perform the ELISA. (55 µL is required if a 1:10 dilution is tested well). 200 µL will leave sufficient plasma for reference (re-testing) purposes, if required. It is generally possible to take greater than 300 µL. Validation studies have shown that IFN-ɣ 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-CMV 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-CMV assay by batching my samples. What is the stability associated with the harvested plasma? 2013 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; plasma kept at -70°C 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 -20What should I do if "clots" form in my plasma samples during their storage at -20°C? C? 2013 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? 2013 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
Human IFN-ɣ° ELISA 2013 The QuantiFERON -CMV assay requires the use of the QuantiFERON -CMI ELISA to measure levels of IFN-ɣ. The procedure detailed in the QuantiFERON -CMV Package Insert for should be followed instead of the Package Insert for the QuantiFERON -CMI ELISA.
Do I test QuantiFERON -CMV plasma samples neat or diluted? 2013 If quantitative measurements of the IFN- ɣ (in IU/mL) are required for all samples, then you need to test the CMV and Mitogen plasmas neat as well as diluted 1:10 in Green Diluent. Therefore you need to allow for 5 ELISA wells per patient being tested. The ELISA is accurate for plasma samples containing up to 10 IU/mL. Use the neat plasma values if below 10 IU/mL, otherwise use the values from the diluted samples (multiplied by 10).
What is the stability associated with: a) Kit Standard? b) Conjugate 100X Concentrate? c) Wash Buffer? 2013 a) Reconstituted IFN-ɣ Standard may be kept for up to 3 months if stored at 2°C to 8°C (the date of reconstitution should be noted). Reconstituted IFN-ɣ standard should be equilibrated at Room Temperature (17°C to 27°C) 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 2°C to 8°C following its use. c) Working strength Wash Buffer may be stored at Room Temperature (17°C to 27°C) for up to 2 weeks.
Can I use the QuantiFERON-CMI ELISA plates immediately after their removal from the fridge? 2013 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? 2013 No. Although an automated plate washer is recommended, manual washing can be performed following the procedure as outlined in the QuantiFERON-CMV Package Insert.
How important is washing during the QuantiFERON-CMI ELISA? 2013 As with most ELISAs, inadequate or incorrect washing is the single most common cause of QuantiFERON-CMI 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? 2013 General ELISA problems with the possible causes and appropriate solutions are listed below. NON-SPECIFIC COLOUR DEVELOPMENT: Possible Cause - Incomplete washing of the plate. Solution - Wash the plate at least 6 times with 400 µL/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. Possible Cause - Cross-contamination of ELISA wells . Solution - Take care pipetting and mixing samples to minimize risk. Possible Cause - Components have expired. Solution - 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. Possible Cause - Components are contaminated Solution - Avoid contamination of components HIGH BACKGROUND: Possible Cause - Incomplete washing of the plate Solution - Wash the plate at least 6 times with 400 µL/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. Possible Cause - Conjugate reconstitution/dilution error Solution - Conjugate 100X Concentrate should be reconstituted with 300 µLof distilled water. Working strength conjugate is prepared by diluting the Conjugate 100X Concentrate 1/100 in Kit Green Diluent as per the Package Insert. Possible Cause - Incubation temperature too high Solution - Incubation of the ELISA should be performed at Room Temperature, 17°C to 27°C. Possible Cause - Components have expired Solution - 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. Possible Cause - Enzyme Substrate is contaminated Solution - Discard substrate if blue coloration exists. Ensure clean reagent reservoirs are used. LOW ABSORBANCE: Possible Cause - Standard Dilution Error Solution - Ensure dilutions of the Kit Standard are prepared correctly as per the Package Insert. Possible Cause - Pipetting error Solution - Ensure pipettes deliver correct volume. Possible Cause - Wash Buffer dilution error Solution - Ensure a 1 in 20 dilution of the wash buffer concentrate is performed to prepare the working strength wash buffer. Possible Cause - Incubation Temperature too low Solution - Incubation of the ELISA should be performed at Room Temperature, 17°C to 27°C. Possible Cause - Incubation time too short Solution - Incubation of the plate with the conjugate, standards and samples should be for 120 +/- 5 minutes. The Enzyme Substrate Solution is incubated on the plate for 30 minutes. Possible Cause - Incorrect Plate Reader filter used Solution - Plate should be read at 450 nm with a reference filter of between 620 and 650 nm. Possible Cause - Reagents are too cold Solution - All reagents, with the exception of the Conjugate Concentrate, must be brought to room temperature prior to commencing the assay. This takes approximately one hour. Possible Cause - Kit / Components have expired. Solution - 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. POOR STANDARD CURVE: Possible Cause - Incomplete washing of the plate Solution - Wash the plate at least 6 times with 400 µL/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. Possible Cause - Standard Dilution Error Solution - Ensure dilutions of the Kit Standard are prepared correctly as per the Package Insert. DUPLICATE VARIABILITY: Possible Cause - Poor Mixing Solution - Mix reagents thoroughly by inversion or gentle vortexing prior to their addition to the plate. Possible Cause - Incomplete washing of the plate Solution - Wash the plate at least 6 times. More than 6 washing cycles may be required depending on the washer being used. A soak time between cycles should be used. Possible Cause - Inconsistent pipetting technique or interruption during assay set-up Solution - Sample and standard addition should be performed in a continuous manner. All reagents should be prepared prior to commencing the assay.
I have very high Nil control values? What may be the problem? 2013 Under most circumstances, the expected IFN-ɣ 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.
How do you convert IU/mL to pg/mL of human IFN-ɣ? 2013 The potency of the International Reference Standard for Human IFN-ɣ is given in IU/mL. Desem and Jones (Clin Diagn Lab Immunol, 5, 531-536, 1998) described 40 pg/mL of human IFN-ɣ as being equivalent to 1 IU/mL.

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