7. QF-PCR FOR RAPID PRENATAL DIAGNOSIS OF ANEUPLOIDY

Previous - Index - Next

This technique is a useful adjunct to prenatal diagnosis and is a more appropriate technique than FISH when dealing with large numbers of prenatal referrals. Internal validation is essential before using this technique. Close liaison with Molecular Genetics colleagues is necessary to establish this technique in a cytogenetic laboratory.

The limitations of QF-PCR in identifying chromosome abnormalities must be clearly known. It is recommended that testing for trisomies 13, 18 and 21 is carried out, whilst it is acceptable to test for sex chromosome aneuploidy in only a subset of referrals. QF-PCR analysis provides information only about the probe locus in question. It does not substitute for a complete chromosome analysis.

7.1 EQUIPMENT, FACILITIES AND SAFETY

The equipment should be regularly maintained and there should be procedures in place for the safe disposal of the waste. The genetic analyser used for the analysis of the STR products should be capable of 2 bp allele resolution and peak area/peak height quantification.

7.2 SAMPLE PREPARATION

For amniotic fluid, between 0.5 and 4 ml or 1/10 of the sample is recommended for QF-PCR analysis, as larger aliquots may compromise the karyotype analysis. For chorionic villus samples, it is recommended that at least two chorionic villi taken from different regions of the biopsy should be processed to minimise the risk of misdiagnosis due to confined placental mosaicism.

A chelex-based method is recommended for DNA preparation as this does not require any tube-tube transfers. Home-made kits should be batch tested using at least a trisomy and a normal DNA sample to ensure consistent assay quality and trisomy diagnosis. A H2O control must also be included in each PCR set-up to identify any DNA or PCR product contamination. Between 24-26 PCR cycles should be carried out as standard practice and a minimum of 4 markers for each chromosome tested to reduce the number of uninformative results. It is recommended that tri/tetra/ penta/hexanucleotide repeat markers are used as these have fewer stutter peaks, although dinucleotide repeat markers are acceptable if few suitable markers are available within the tested region.

New markers not used previously for QF-PCR aneuploidy diagnosis should be validated by testing a minimum of 100 chromosomes, including aneuploid samples.

7.3 ANALYSIS

It is recommended that both the electrophoretogram and peak measurements, which can be transferred to a spreadsheet for convenience, are analysed. To ensure the quality of the data both minimum and maximum peak heights should be used. It is acceptable to fail individual markers if there are valid technical reasons such as bleedthrough between colours and electrophoretic spikes. It is acceptable to use peak height, peak area or both measurements to calculate allele ratios, although for results obtained from an automated sequencer it is recommended that peak area is used to minimise peak distortion due to widely-spaced alleles.

The area/height of the shorter length allele should be divided by that of the longer length allele and the normal range should not exceed 0.8-1.4.

To interpret a result as abnormal, at least two informative marker results should be consistent with a triallelic genotype, with all the other markers uninformative. It is unacceptable to interpret a result as abnormal if shown by only one marker. Confirmation of sample identity when a result is abnormal by repeat PCR of the DNA, re-extraction of samples, or mat blood analysis is recommended.

To interpret a result as normal at least two informative marker results consistent with a normal diallelic pattern are required, with all other markers uninformative. However, it is acceptable to report single marker results that have a normal diallelic pattern and all other markers uninformative as consistent with a normal chromosome complement, if the report states that the result is based on a single marker result and that this the result must be confirmed.

Where maternal cell contamination occurs, if allele ratios are inconclusive and/or the maternal genotype is present at a high level it is recommended that the fetal genotype should not be interpreted.

7.4 REPORTING

It is recommended that the assumption that fetal material is tested, and the fact that mosaicism and small segment imbalance for chromosomes tested may not be detected should be included on the report, either in the main text or in a report rider. The locations of markers showing a triallelic result should be listed to define the trisomic region. It is acceptable to list markers on a normal report, although this should be done in a way that does not 'bury' the result.

It is acceptable to report normal QF-PCR results as 'consistent with a normal diploid complement for chromosomes 13, 18 and 21', 'an apparently normal complement of chromosomes 13, 18 and 21 was detected', 'no evidence of trisomy' or similar statement.

Abnormal reports should include an interpretative statement such as 'consistent with Down syndrome', 'associated with Down syndrome', 'indicative of Down syndrome' or 'predicted to be affected with Down syndrome'. It is important to be aware that the QF-PCR sex chromosome assay (Donaghue et al. 2003) is a highly stringent screen for monosomy X but NOT a diagnostic test. A result consistent with monosomy X, where all polymorphic markers have only a single allele peak and no Y sequences are present, may represent a normal female homozygous for all markers tested. Therefore such a result should either be confirmed using another technique, or reported as being consistent with monosomy X with the caveat that there remains a possibility that a normal female could give the same genotype.

The limitations of the chromosome analysis or FISH probe being used must be clearly known. FISH analysis provides information only about the probe locus in question. It does not substitute for a complete chromosome analysis.

Care must be taken in the interpretation of normal results from studies based on repeated sequence probes, due to rare individuals with small numbers of the target repeated sequence.

Interpretation of results requires supervision by an appropriately trained cytogeneticist or physician.

Previous - Index - Next

Last changed: 2008-02-05