Rapid Prenatal Diagnosis
What is QFPCR?
QFPCR stands for quantitative fluorescence polymerase chain reaction. Small sections (markers) of DNA from the sample are amplified, labelled with fluorescent tags and the amounts measured by electrophoresis.
What can it test for?
QFPCR is used to test for gene dosage – ie. the number of copies of a given gene present in a sample. The YNEGLH Central Lab uses QFPCR to test for aneuploidy of whole chromosomes – currently chromosomes 13, 18 and 21 – in amniotic fluid samples. It is possible to test for sex chromosomes by QFPCR but the Leeds lab does not currently offer this service.
What do the results look like?
The results are presented as a graph and ratios are calculated using a spreadsheet.
For each marker tested, a normal result is represented on the graph as two peaks of equal height and area (one peak for each chromosome present). So, for example, a normal pattern for the chromosome 13 marker D13S742 would look like this:
The name of the marker is given in the grey box above the peaks. The numbers underneath the peaks represent the length of the marker (sz) and the amount present (peak area, ar). So this sample has one D13S742 marker which is 248.01 bases long and another which is 280.83 bases long. The two copies are referred to as ‘alleles’ and there is one on each copy of chromosome 13, present in a ratio of 13200:12550 or approximately 1:1.
An example trace for a normal sample is shown below.
The four rows on the graph correspond to the four different fluorescent tags used to label the DNA – at least one marker per chromosome is present on each row, with five markers per chromosome in total.
In this example, the chromosome 21 marker D21S311 has only a single peak visible. This is because it has two alleles of the same length and the two peaks are superimposed. This means that a ratio cannot be calculated and the marker is uninformative.
If the sample is abnormal, a different pattern is seen on the trace:
Markers D21S11 and D21S1411 show three peaks of equal area, indicating that there are three different alleles present in a 1:1:1 ratio. Marker D21S1435 shows only two peaks but the ratio of the peak area for the two alleles is 2:1. The 2:1 ratio also indicates the presence of three alleles, however two of the alleles are of the same length, and therefore superimposed as for marker D21S11 in the ‘normal’ example.
All of these results indicate the presence of three copies of chromosome 21, meaning that the foetus will have Down syndrome. Note that the chromosome 13 and chromosome 18 markers show a normal pattern.
Why are you testing by QFPCR and not FISH?
FISH is a very labour-intensive method. Testing by QFPCR instead means that we can test samples more efficiently. There is no difference between the two techniques in either the success rate or the quality of the information obtained.
Another advantage of QFPCR is that, if necessary, we can positively identify samples by analysis of maternal blood. This is not possible by FISH. Additionally, we can detect any maternal cell contamination of the sample.
Are there any disadvantages?
QFPCR may not detect low level mosaicism for an abnormal cell line (<15 – 20% abnormal cells).
QFPCR is not a reliable method for the detection of deletions.
In rare cases, samples may be uninformative for all the markers on one or more chromosomes. Such cases will be tested by alternative methods (FISH or full karyotyping).
Some samples may produce ambiguous results which will need further investigation.
Although the majority of these will be normal, this process may delay the production of results by 1 or more days.
What are the sample requirements?
10-15ml of amniotic fluid, clearly labelled with at least two patient identifiers, should be sent to the lab, together with a sample of maternal blood (5-10 ml in EDTA). The maternal blood sample will be used in some cases to clarify the result; unused maternal bloods will be discarded once the PCR result has been issued.
All amniotic fluid samples will be tested by QFPCR, with a subset of pregnancies also being tested by full karyotyping if there is a clear clinical indication (eg. abnormal ultrasound scan).
What if the sample is bloodstained?
The lab will attempt a QFPCR test on all samples, even if they are bloodstained. However, if the blood is of maternal origin, the sample may be unsuitable for analysis because the maternal signals interfere with the foetal ones. Samples in this category will be tested by full karyotyping, irrespective of the reason for referral.
How soon will I get my results?
The lab aims to report all samples on the working day after the sample was received. Samples received before 3pm will be set up the same day; those received after 3pm will be set up the following day.
The lab will telephone results to the requesting clinician or screening coordinator as soon as they are available.
If further testing is required due to an ambiguous or uninformative result, the final report may be delayed by 1 or more days.