Most markers included in Aneufast™ have been extensively validated and applied on over 25.000 clinical specimens.
Why QF-PCR for Rapid Prenatal Diagnosis of Commom Aneuploides.
Advantages of QF-PCR over FISH for the prenatal diagnosis lab.
FISH analysis requires counting fluorescent signals on interphase nuclei; the interpretation is not always clear-cut.
For this reason, counting several cells for each chromosome specific probe is strongly recommended.
Interpretation may also be subjective requiring independent analyses from two operators. This makes the test time consuming, labour intensive and costly.
Furthermore the test is not prone to automation so that high throughput of samples is not allowed; all the above mentioned have restricted FISH application to selected high risk pregnancies.
QF-PCR interpretation is usually straightforward.
Results are obtained by a simple calculation of numerical value and it is totally objective.
All steps of the procedure are easy to automate and very high throughput is possible, this greatly reduces overall costs.
The turnaround time can be less than 4 hours in urgent cases.
PCR amplification with fluorescent primers is by far more sensitive than hybridisation and very little volume of amniotic fluid (0,5 ml instead of 5ml) is needed to perform the test. This makes rapid diagnosis also possible in difficult cases where only small sample volumes are obtained without compromising the success of cell culture.
By keeping small aliquots of all samples as back up, it is also possible to perform the test in cases were rapid diagnosis was not previously planned such as slow growing cultures or complete culture failures.
QF-PCR analysis is accurate on a variety of samples such as amniotic fluid, CVS, fetal, neonatal, or adult blood, buccal cells, cell cultures, fixed cells and tissues.
Advantages of QF-PCR over FISH for Healthcare professionals.
Speed and automation together with low cost allow offering the test to all prenatal samples as a preliminary to conventional cytogenetics.
QF-PCR report is usually issued within 24 hours from sampling; urgent cases can be processed in less than 4 hours. This allows prompt reliefs of anxiety in both parents while waiting for completion of cytogenetic analysis or improves pregnancy management in cases of abnormal results.
Aneufast always includes primers for the most common aneuploidies so that it is not necessary to choose few probes depending on the indications to reduce the cost.
The efficiency is not influenced by the gestational age and the high rate of uninformative samples reported for FISH analysis late in pregnancy has never been observed by QF-PCR.
Highly polymorphic microsatellite markers are amplified to perform the diagnosis; these can provide additional information of great clinical value such as:
The origin of the extra chromosome in trisomic samples and the meiotic phase were non disjunction occurred.
The zygosity in multiple pregnancies, independently from chorionicity and fetal sex.
Microsatellites can also allow performing diagnosis on samples suspected of being contaminated by maternal cells, such as heavy bloodstained amniotic fluids, without any risk of misdiagnosis. In these cases the analysis of a maternal sample (mouthwash or swab) easily allows distinguishing maternal and fetal DNA even in female fetuses
Markers are selected along each of the examined chromosomes increasing the likelihood of also detecting chromosome rearrangements (such as isochromosomes) resulting in partial trisomies.
How is prenatal diagnosis by QF-PCR is performed?
A small aliquot of fetal sample (i.e. 0.5-1mL amniotic fluid, 5µL blood) is used for DNA extraction and molecular diagnosis. This will not affect culturing the samples for conventional cytogenetic analysis.
After DNA is obtained, selected highly polymorphic markers are amplified by the Quantitative Fluorescent PCR assay, at least four markers on each chromosome 21, 18, 13, X and Y are simultaneously evaluated. Products are than analysed by capillary electrophoresis on an automated DNA sequencer.
The whole laboratory procedure takes under 4 hours to be completed.
How accurate is rapid prenatal diagnosis by QF-PCR?
QF-PCR is so accurate that in some cases it is used as a stand-alone prenatal diagnostic test.
The QF-PCR assay has already been applied on over 50.000 prenatal samples in different genetic centres over the last 5 years; false positive or negative results have never been reported.
QF-PCR has repeatedly demonstrated 100% sensitivity and specificity in detecting common autosomal trisomies. On a large cohort of 18000 clinical cases, QF-PCR has been shown able to detect 95% of all clinically significant abnormal karyotypes in a few hours after sampling.