Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR‐based genetic analysis

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.