affigene General
Q: Can I use an affigene® trender kit as a qualitative test?
A: A titre given by the affigene® analysis software means the sample is positive. The affigene trender kits have been designed for monitoring patients to determine changes in titer levels.
Q: Why are there different values for the calibrators when using plasma or whole blood in affigene® CMV and EBV trender kit?
A: There are different values for the calibrators using plasma or whole blood because the DNA yield from sample preparation is different from plasma and whole blood. Calibration of the calibrators is done for plasma and whole blood separately.
Q: What is the Precision for affigene® trender assays?
A: The variation run-to-run is about ±0,2 log. A sample with a titre of 4 log may vary between 3,8 to 4,2 log. See the User Manuals, section performance characteristics for more information.
Q: What are the positive controls or high and low calibrators made from?
A: The PC and Calibrators are PCR fragments of the target, made from a plasmid. They are stored in a Tris buffer.
Q: Against what quality controls or standards do you calibrate BKV kits?
A: We have calibrated the BKV calibrators against well characterised and quantitated standard material (serotype I (genotype DUN)) from the Swedish Center for Disease Control.
Q: Are there any toxic components in the affigene® trender-tracer kits?
A: No, there are no toxic substances in the affigene® trender/tracer kits. There are MSDS documents (Material Safety Data Sheet) for each product, which can be supplied from Customer Service.
Q: What is a scorpion primer?
A: Scorpion primers are structurally and functionally related to molecular beacons, but serve also as probes in the PCR reaction. Scorpion primers have self-complementary sequences that form a 5´-terminal stem-loop structure, with the loop sequence complementary to the amplicon sequence, which follows the primer sequence. The 3´-end serves as the probe. The stem region is labelled with a reporter fluorophor and a quencher.
In the first step, the primer is extended, yielding a single-stranded template for the reverse primer in the second step. The stem then opens and the loop binds to the product, separating reporter and quencher.
The reverse extension is blocked by a hexethylene glycol group. This ensures that the reporter of the scorpion probe remains quenched in unspecific products (e.g. primer dimmers).ScorpionsTM is a trademark of DxS.
Q: affigene HSV 1/2 utilizes CSF and swab samples for determination in real-time PCRIs there a protocol that utilizes serum or plasma samples?
A: All validations have been done on CSF and swabs, both cell-free matrices. As serum and plasma are also cell-free matrices there is no reason to believe these matrices would not work, but there is no performance data for these matrices.
Q: What is the quantitative range for HBV trender using serum samples?
A: The quantitative range for serum samples is 172-1,72E8 IU/mL. A precision study is performed which shows the same precision for plasma and serum within the quantitative range. Remember to always use the same sample matrix throughout the monitoring of each patient.
Q: Can I compare copies/mL and copies/100 000 cells?
A: 1 mL whole blood corresponds to about 7 500 000 cells. For more information, see User Manual for Performance characteristics, LOD.
Q: Which part of the EBV genome is amplified
A: A part of the EBNA-1 gene.
Q: What is a positive sample?
A: For the affigene® trender assay a titer given by the affigene® analysis software is a positive sample.
For the affigene® tracer assay a sample reported as "detected" in the affigene® analysis software is a positive sample.
Q: What is the difference between linear range, dynamic range and quantitative range?
A: There is some confusion about the definitions of the dynamic range, linear range and quantitative range of an assay. For some manufacturers/users all these terms are used meaning the same thing, sometimes they define completely different ranges. At SMD we follow the NCCLS guidelines in terminology and study design. Our interpretation of the terms is described below.
The performance characteristics of an assay depend on:
- How the sample is prepared, e.g. which input and output volumes are used (concentration factor), removal of inhibitors as well as the DNA/RNA recovery in sample preparation.Sample preparation accounts for a considerable portion of the imprecision of the system.
- Robustness of the PCR system.
Note: To obtain performance characteristics data for the complete assay sample preparation has to be included in the performance studies. If PCR fragments (e.g. a DNA quantitation standard) are used in the study performance data are only generated for the PCR system alone. These performance data are not comparable with performance data for the complet assay i.e including sample preparation. "Real" samples are always clinical.
Q: What is limit of detection (LOD)?
A: OD is defined as the titre level where 95% of all replicates of a sample are determined positive. The LOD level is usually determined by probit analysis, resulting in a number together with a range (95% confidence interval), which should always be specified. Note: Results below LOD are still true positives, but less than 95% of all replicates may be determined positive.
Example: Consider a fictive assay with LOD 200 copie/mL. At the expected titer 200 copies/mL 95% of the replicates will be reported positive.
At 60copies/mL some 50% of the replicates will be reported positive. At 20 copies/mL maybe 20% of the replicates will be reported positive. The LOD of an assay depends on:
- Sample preparation, input and output volumes (concentration factor)
- Robustness of the PCR system. The ability to amplify one single copy of prepared DNA target.
Note: Clinical material should be used, see above.
Q: What is precision?
A: Precision is defined as closeness of agreement between independent test results and is expressed quantitatively in terms of imprecision (e.g. standard deviation). In affigene® assays the precision is determined with maximum variation in parameters such as operators, pipettes and test occations.
Q: What is accuracy?
A: Accuracy is defined as agreement between obtained result and true value, e.g. international reference standards.
Q: What is Quantitative range?
A: The quantitative range is the range where the assay fulfils claimed precision as well as accuracy.
Note: Samples with titers below the quantitative range are still true positives, but precision has not been determined.
Q: What is Linearity/ linear range?
A: The range where the assay is linear. Linear range is not a relevant term in real-time PCR since the technology as such usually is linear even below the limit of detection. Neither precision nor accuracy needs to be fulfilled. Linear range is not used in NCCLS guidelines or by SMD.
Q: What is Dynamic range?
A: This term is used by some manufacturers. There is no clear definition of the term and no recommendations by the NCCLS guidelines to use it. The complete range from LOD to the upper limit in the quantitative range is often referred to as dynamic range. In contrast to quantitative range no precision or accuracy is guaranteed and therefore a wider range is obtained.
Note: Samples below the dynamic range are still true positives.
Q: Are your products CE-labelled?
A: All our products are CE-labelled.
Q: Do you have a license for PCR?
A: Yes, we have a world-wide license for PCR for in vitro diagnostics, including realtime PCR.
Q: Is the CE-label valid when using a non-CE labelled PCR machine?
A: Yes, affigene® trender has been validated and CE-labelled for use with Stratagene Mx3000P and other general purpose instruments according to User Manual.
Q: The sample titer obtained with affigene® trender does not correspond to results obtained using my previous protocol?
A: Unit. Make sure that the unit used for sample reporting is comparable (c/mL, gEq/mL, IU/ml etc.)
Calibration. Make sure that the quantitative standards are calibrated to match relevant international standards (WHO, QCMD, VQC etc). All affigene® trender assays are calibrated to match available international standards.
Performance characteristics. Sample titers may differ due to variation in the method itself. All quantitative CE-labelled IVD devices should declare precision and linearity data to comply with the IVE-directive. Verify whether titer difference is within the variance of the respective assays.
Number of samples/replicates. Verify that the number of samples/replicates are significant. Direct comparison of few samples/replicates does not give the whole picture.
Q: How can I compare my results with results from another laboratory?
A: Our products are standardized to ensure comparability of results between different sites.
Q: Are two calibrators enough to give a reliable sample titer?
A: Yes, The affigene® assays have been completely validated and CE-approved using two calibrators. During product development we use 7-10 standard points. Later in the development process two of these points are chosen. These points are chosen near the extremes of the quantitative range (one near the lower end and one near the higher end). Then criteria are established which guarantee that PCR efficiency and linearity are as good as it would have been with all seven standard points. This means better economy (and faster results) for the lab as five more positions can be used for clinical samples.
Q: Is the test able to monitor only EBV reactivations or primary infections?
A: The affigene EBV trender kit has been designed for monitoring immuno compromised patients such as transplant recipients. It can be used for both monitoring patients (reactivation) and for detection of primary infections.
Q: Is it possible to use urine-samples in affigene® CMV trender kit?
A: The affigene® CMV trender kit is CE-laballed for plasma and whole blood samples. Other applications can be developed on request
Q: Which part of the CMV genome is amplified?
A: Major Immediate Early gene.
Q: How can I be sure to pick up different variants of CMV?
A: CMV trender is developed to detect all known CMV variants.
Q: What is the IC (internal control) manufactured from?
A: The internal control is synthesized from non-viral, non-bacterial, non-human DNA.