Automatic scanning was more efficient and consistent than manual scanning for detection of rare FCs and required considerably less operator time. Automatic scanning using FISH is more sensitive than that using PRINS. The study validates automatic scanning retrieval of FCs from maternal blood.
Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1–10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning.
Data obtained raise questions about the appropriateness of the DGC step for the enrichment of rare FCs and argues for the use of the alternative nonaggressive version of the procedure presented here or prioritizing other methods of enrichments.
Objectives: Analysis of DNA from small numbers of cells, such as fetal cells in maternal blood, is a major limiting factor for their use in clinical applications. Traditional methods of single-cells whole genome amplification (SCs-WGA) and accurate analysis have been challenging to date. Our purpose was to assess the feasibility of using a few fetal cells to determine fetal sex and major chromosomal abnormalities by quantitative fluorescent polymerase chain reaction (QF-PCR). Methods: Cultured cells from 26 amniotic fluid samples were used for standard DNA extraction and recovery of 5 fetal cells by laser-capture microdissection. SCs-WGA was performed using the DNA from the microdissected cells. PCR amplification of short tandem repeats specific for chromosomes 13, 18, 21, X and Y was performed on extracted and amplified DNA. Allele dosage and sexing were quantitatively analyzed following separation by capillary electrophoresis. Results: Microsatellite QF-PCR analysis showed high concordance in chromosomal copy number between extracted and amplified DNA when 5 or more cells were used. Results were in concordance with that of conventional cytogenetic analysis. Conclusion: Satisfactory genomic coverage can be obtained from SCs-WGA. Clinically, SCs-WGA coupled with QF-PCR can provide a reliable, accurate, rapid and cost-effective method for detection of major fetal chromosome abnormalities.
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