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Some caveats in PCR-based prenatal diagnosis on direct amniotic fluid versus cultured amniocytes
R M Frederickson1, H S Wang, L C Surh
1Department of Clinical Chemistry, University of Ottawa, Ontario, Canada.
Abstract:
The polymerase chain reaction (PCR) offers new advances in prenatal genetic diagnosis particularly with limitations in amount of sample, turn-around time of results, and costs. However, maternal contamination is a concern in any fetal sampling, and even more so with PCR given its potential to detect at the level of a few cells. We report our experience with 53 matched pairs of direct and cultured amniocytes using three independent DNA markers amplified by PCR within the setting of a service molecular diagnostic laboratory. Despite 15/53 (30 per cent) of the amniotic fluids showing visible red blood cells prior to culturing, only 5/53 (9 per cent) showed trace PCR contamination. Of note, this was found on only one marker with a particularly robust PCR product of small size and at such a low level that it was unlikely to have resulted in ambiguous interpretation. One of the cultures also showed a similar type of contamination with this same marker. However, in addition, there were 2/53 (3.7 per cent) cultures which showed substantial maternal contamination detected by all three PCR markers, but not visualized on the originating direct samples. Our results suggest that the careful use of direct amniocytes for molecular genetic testing by PCR is reliable and reproducible in most cases.
Insights
Direct amniocyte use in prenatal genetic testing via polymerase chain reaction (PCR) is reliable. Careful application minimizes maternal contamination risks, ensuring accurate fetal DNA analysis.
Area of Science:
- Molecular Biology
- Genetics
- Prenatal Diagnostics
Background:
- Polymerase chain reaction (PCR) offers advancements in prenatal genetic diagnosis, addressing limitations in sample quantity, turnaround time, and cost.
- Maternal contamination is a significant concern in fetal cell sampling, especially with PCR's high sensitivity.
Purpose of the Study:
- To evaluate the reliability and reproducibility of using direct amniocytes for prenatal genetic testing with PCR.
- To assess the incidence and impact of maternal contamination in PCR-based prenatal diagnostics.
Main Methods:
- Analysis of 53 matched pairs of direct and cultured amniocytes.
- Amplification of three independent DNA markers using PCR.
- Comparison of contamination levels between direct samples and cultured cells.
Main Results:
- Only 9% of direct amniotic fluid samples showed trace PCR contamination, primarily on a single, robust marker at low levels.
- 3.7% of cultured amniotic fluid samples exhibited substantial maternal contamination across all three markers, not evident in direct samples.
- Visible red blood cells in 30% of amniotic fluids did not consistently correlate with PCR contamination.
Conclusions:
- Direct amniocytes are a reliable source for PCR-based molecular genetic testing in prenatal diagnosis.
- Careful laboratory practices can effectively manage and minimize maternal contamination risks.
- Cultured amniocytes may present a higher risk of undetected maternal contamination compared to direct samples.