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Detection of maternal cell contamination in amniotic fluid cell cultures using fluorescent labelled microsatellites
G W Smith1, C A Graham, J Nevin
1Department of Medical Genetics, Belfast City Hospital, Northern Ireland.
Abstract:
A rapid PCR based assay was used to ascertain the presence of maternal cell contamination (MCC) in amniotic fluid cell cultures and to exclude MCC in cases where cytogenetic analysis was possible only from one primary cell culture. Six 6-carboxyfluorescein (FAM) and three 6-carboxyfluorescein hexachloride (HEX) labelled primer sets were used to amplify two tetra- and seven dinucleotide repeat polymorphisms. The PCR amplifications were multiplexed in (three) three primer set reactions and visualised on an Applied Biosystems 373A sequencer running Genescan 672 software. The microsatellite products obtained from 200 amniotic fluid cell cultures where the karyotype was female were compared against corresponding maternal blood PCR products. A single case of MCC was detected indicating the usefulness of such assays. We suggest that screening for MCC should be considered in instances where the amniotic fluid sample is bloodstained or was obtained with difficulty, or where the karyotype is female and chromosome analysis is not possible from more than one primary cell culture.
Insights
A new PCR assay effectively detects maternal cell contamination (MCC) in amniotic fluid cultures. This method is crucial for ensuring accurate genetic testing, especially when only one cell culture is available.
Area of Science:
- Molecular Biology
- Genetics
- Prenatal Diagnostics
Background:
- Maternal cell contamination (MCC) can compromise the accuracy of prenatal genetic analyses performed on amniotic fluid.
- Cytogenetic analysis of amniotic fluid is sometimes limited to a single primary cell culture, increasing the risk of misdiagnosis due to MCC.
Purpose of the Study:
- To develop and validate a rapid Polymerase Chain Reaction (PCR)-based assay for detecting maternal cell contamination (MCC) in amniotic fluid cell cultures.
- To assess the utility of this assay in cases where cytogenetic analysis is feasible from only one primary cell culture.
Main Methods:
- Utilized six 6-carboxyfluorescein (FAM) and three 6-carboxyfluorescein hexachloride (HEX) labeled primer sets for PCR amplification.
- Targeted two tetra- and seven dinucleotide repeat polymorphisms, multiplexing PCR reactions into three primer set combinations.
- Analyzed PCR products using an Applied Biosystems 373A sequencer with Genescan 672 software.
- Compared microsatellite profiles from 200 female amniotic fluid cell cultures with corresponding maternal blood samples.
Main Results:
- Successfully detected a single instance of maternal cell contamination (MCC) among the 200 samples analyzed.
- Demonstrated the effectiveness of the PCR-based assay in identifying MCC.
Conclusions:
- The developed PCR assay is a valuable tool for detecting maternal cell contamination in amniotic fluid.
- Screening for MCC is recommended for bloodstained amniotic fluid samples, samples obtained with difficulty, or when female karyotype analysis is limited to one primary cell culture.