Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR-based genetic analysis |
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Authors: | J. Büsch P. Huber E. Pflüger St. Miltenyi J. Holtz Professor Dr. A. Radbruch |
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Affiliation: | 1. Institute for Forensic Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany;2. Miltenyi Biotec GmbH, Friedrich-Ebert-Str., 51429 Bergisch Gladbach, Germany |
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Abstract: | For simple and effective isolation of fetal cells from peripheral maternal blood, we combined depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell separation (MACS). First CD45+ and CD14+ cells were depleted from maternal peripheral blood mononuclear cells by MACS. From the depleted fraction, CD71+ erythroid cells were enriched up to 80 per cent by MACS. This ‘double-MACS’ procedure yielded an average depletion rate of 780-fold and an average enrichment rate of 500-fold, with approximate recovery rates of 40–55 per cent. For paternity testing, cells from unseparated blood and the various fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45−/CD71+ sorted cells from maternal blood, but not in unfractionated cells from maternal blood or CD45−/CD14− cells, paternal alleles could be detected. In the CD45−/CD71+ fraction, the relative frequency of paternal alleles compared with maternal alleles ranged from 1 in 20 to 1 in 200 (determined by titration and depending on the quality of separation and biological variation). In 7 out of 11 cases, between weeks 12 and 25 of gestation, we could identify paternal alleles by PCR, either HLA-DQ-A1 or D1S80. This double-MACS procedure is simple, fast, efficient, and reliable for non-invasive prenatal diagnosis. |
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Keywords: | Prenatal paternity testing high-gradient magnetic cell separation MACS primer extension preamplification HLA-DQ-A1 D1S80 CD71 |
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