QuntiFERON – TB Gold Plus (QFT-Plus)
Introduction
The tuberculin skin test (TST) has limitations for latent tuberculosis infection (LTBI) diagnosis in low-prevalence settings. Previously, all TST-positive individuals referred from the community to Baltimore City Health Department (BCHD) were offered LTBI treatment, after active TB was excluded. In 2010, BCHD introduced adjunctive QuantiFERON-TB Gold In-Tube (QFT-GIT) testing for TST-positive referrals. We evaluated costs and cost-effectiveness of this new diagnostic algorithm.
Basic Principles
The QFT-Plus assay uses specialized blood collection tubes, containing peptide antigens that simulate M. tuberculosis proteins, which are used to collect whole blood. Incubation of the blood occurs in the tubes for 16 to 24 hours, after which, plasma is harvested and tested for the presence of IFN-γ produced in response to the peptide antigens.
The QFT-Plus test is performed in two stages. First, whole blood is collected into each of the
QFT-Plus Blood Collection Tubes, which include a Nil tube, TB1 tube, TB2 tube and a Mitogen tube. Alternatively, blood may be collected in a single blood collection tube that contains lithium-heparin as the anticoagulant, and then transferred to QFT-Plus Blood Collection Tubes.
Specimen Collection and Handling
a. QuantiFERON Nil tubes (gray cap with white ring; use between sea level
b. QuantiFERON TB1 tubes (green cap with white ring; use between sea level
c. QuantiFERON TB2 tubes (yellow cap with white ring; use between sea level
d. QuantiFERON Mitogen tubes (purple cap with white ring; use between sea level
Antigens have been dried onto the inner wall of the blood collection tubes, so it is essential that the contents of the tubes be thoroughly mixed with the blood. For blood directly drawn into the QFT-Plus Blood Collection Tubes, the QFT-Plus Blood Collection Tubes must be transferred to a 37°C incubator as soon as possible and within 16 hours of collection. Alternatively, blood may be collected into a single lithium-heparin tube for storage prior to transfer to QFT-Plus Blood Collection Tubes and incubation. Blood specimens collected in lithium-heparin tubes can be stored up to 12 hours at room temperature (17–25°C) followed by transfer to QFT-Plus Blood Collection Tubes, or blood specimens in lithium-heparin tubes can be transferred to QFT-Plus Blood Collection Tubes directly after collection. Blood specimens in lithium-heparin tubes may also be stored at 2–8 °C for 16 to 48 hours prior to transfer to the QFT-Plus Blood Collection Tubes.
Post-incubation of blood collection tubes and harvesting of plasma
a. Prior to harvesting plasma, samples in QFT-Plus Blood Collection Tubes must have been incubated at 37°C for 16–24 hours.
b. After incubation of the tubes at 37°C ± 1°C, harvesting of the plasma is facilitated by centrifuging tubes for 15 minutes at 2000 to 3000 RPM. The gel plug will separate the cells from the plasma.
c. Plasma samples can be stored in centrifuged QFT-Plus Blood Collection Tubes for up to 28 days at 2°C to 8°C. Or harvested plasma samples can be stored for up to 28 days at 2°C to 8°C. Harvested plasma samples can also be stored below –20°C (preferably less than –70°C) for extended periods.
Important: Blood collection tubes (QFT-Plus Blood Collection Tubes for direct draw, or lithium heparin Tubes when blood is collected into a single lithium-heparin tube initially) should be at room temperature (17–25°C/62.6 –77°F) at the time of blood collection.
Requirement
1. Wash buffer 100 ml for 2 liter distilled water
2. Substrate solution
3. Stop solution
4. Green diluents (GD) - for sample diluents
5. Inferno – standard (dissolve 2.27 ml in distilled water)
6. Conjugate 100x concreted (dissolved in 0.3 ml in distilled water)
7. Microplate strips
8. Centrifuge (2000 to 3000 RPM for 15 minutes)
Sample Labeling
Ø 1/185 (1 is sample start number, 185 is Bach run number)
Sample by color coding – Blood sample should be until black mark.
a. Gray = 1N (1 is serial no., N is nil)
b. Green = 1T1 (1 is serial no., T for Test 1)
c. Yellow = 1T2 (1 is serial no., T for Test 2)
d. Purple = 1M (1 is serial no., Mitogen 1)
Step – 1 Preparation of standers
a. Take 4 test tubes and label: S1, S2, S3, S4
b. Add 300 μl of GD to S1, S2, S3, S4
c. Add 300 μl of the kit standard to S1 and mix thoroughly
d. Transfer 100 μl from S1 to S2 and mix thoroughly
a. Transfer 100 μl from S2 to S3 and mix thoroughly
b. GD alone serves as the zero standard (S4)
Step – 2 Preparation of conjugate
Number of strips | Volume of conjugate (100x concentrate) | Volume of Green Diluent |
2 | 10 μl | 1.0 ml |
3 | 15 μl | 1.5 ml |
4 | 20 μl | 2.0 ml |
5 | 25 μl | 2.5 ml |
6 | 30 μl | 3.0 ml |
7 | 35 μl | 3.5 ml |
8 | 40 μl | 4.0 ml |
9 | 45 μl | 4.5 ml |
10 | 50 μl | 5.0 ml |
Step – 3
ü Sample loading in ETI Max 3000
ü Follow up machine
ü Wait 3 to 5 hrs for plate reading
Step – 4 Open software QFT Plus
Ø Run - day and date
Ø Run – number
Ø Kit Bach number – Kit Lot Number
Ø Operator Name - ----------------------
Step – 5 Reading Write in Colum
 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A | 1 N | 3 N | 5 N | 7 N | 9 N | S1 | S1 | 13 N | 15 N | 17 N | 19 N | 21 N |
B | 1 TB1 | 3 TB1 | 5 TB1 | 7 TB1 | 9 TB1 | S2 | S2 | 13 TB1 | 15 TB1 | 17 TB1 | 19 TB1 | 21 TB1 |
C | 1 TB2 | 3 TB2 | 5 TB2 | 7 TB2 | 9 TB2 | S3 | S3 | 13 TB2 | 15 TB2 | 17 TB2 | 19 TB2 | 21 TB2 |
D | 1 M | 3 M | 5 M | 7 M | 9 M | S4 | S4 | 13 M | 15 M | 17 M | 19 M | 21 M |
E | 2 N | 4 N | 6 N | 8 N | 10 N | 11 N | 12 N | 14 N | 16 N | 18 N | 20 N | 22 N |
F | 2 TB1 | 4 TB1 | 6 TB1 | 8 TB1 | 10 TB1 | 11 TB1 | 12 TB1 | 14 TB1 | 16 TB1 | 18 TB1 | 20 TB1 | 22 TB1 |
G | 2 TB2 | 4 TB2 | 6 TB2 | 8 TB2 | 10 TB2 | 11 TB2 | 12 TB2 | 14 TB2 | 16 TB2 | 18 TB2 | 20 TB2 | 22 TB2 |
H | 2 M | 4 M | 6 M | 8 M | 10 M | 11 M | 12 M | 14 M | 16 M | 18 M | 20 M | 22 M |
Step – 6 Standard Reading
QFT-Plus specificity study results by TB antigen tube
Interpretation based on TB | ||||
Antigen-Nil | QFT-Plus (positive by | Concordant positive TB1 and | ||
IU/ml in | TB1 | TB2 | TB1 and/or TB2)* | TB2 (alternate analysis)† |
Positive | 10 | 18 | 20 | 8 |
Negative | 723 | 715 | 713 | 725 |
Indeterminate | 0 | 0 | 0 | 0 |
97.3% | ||||
Specificity (95% CI) | – | – | (713/733) | – |
(95.8–98.2) | ||||
98.6% | 97.5% | 98.9% | ||
Negativity rate (95% CI) | (723/733) | (715/733) | – | (725/733) |
(97.5–99.3) | (96.2–98.4) | (97.9–99.5) |
_________________________________________________________________________________
* Interpretation based on a TB antigen – Nil value ≥0.35 IU/ml in both (TB1 and TB2) or either TB tube to fit the interpretation criteria for the QFT-Plus (TB1 or TB2) to be determined positive.
* Interpretation based on a TB antigen – Nil value ≥0.35 IU/ml in both (TB1 and TB2) or either TB tube to fit the interpretation criteria for the QFT-Plus (TB1 or TB2) to be determined positive.
† Alternate analysis provided for information only.
Interpretation of results
QFT-Plus results are interpreted using the following criteria.
Important: Diagnosing or excluding tuberculosis disease, and assessing the probability of LTBI, requires a combination of epidemiological, historical, medical, and diagnostic findings that should be taken into account when interpreting QFT-Plus results. See general guidance on the diagnosis and treatment of TB disease and LTBI
Interpretation of QFT-Plus test results
Nil (IU/ml) | TB1 minus Nil | TB2 minus Nil | Mitogen minus | QFT-Plus Result Report/interpretation | ||
(IU/ml) | (IU/ml) | Nil (IU/ml)* | ||||
≤8.0 | ≥0.35 and | Any | ||||
≥25% of Nil | M. tuberculosis | |||||
Any | Positive† | |||||
Any | ≥0.35 and | infection likely | ||||
≥25% of Nil | ||||||
<0.35 or | <0.35 or | M. tuberculosis | ||||
≥0.35 and | ≥0.35 and | ≥0.50 | Negative | |||
infection NOT likely | ||||||
<25% of Nil | <25% of Nil | |||||
<0.35 or | <0.35 or | Likelihood of | ||||
≥0.35 and | ≥0.35 and | <0.50 | ||||
Indeterminate‡ | M. tuberculosis | |||||
<25% of Nil | <25% of Nil | |||||
infection cannot be | ||||||
>8.0§ | Any | determined | ||||
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