ENRICHMENT OF FETAL CELLS FROM MATERNAL BLOOD BY HIGH-GRADIENT MAGNETIC CELL SORTING (DOUBLE MACS) FOR PCR-BASED GENETIC-ANALYSIS

For simple and effective isolation of fetal cells from peripheral mate rnal blood, we combined depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell separation (MACS). First CD 45(+) and CD14(+) cells were depleted from maternal peripheral blood m ononuclear cells by MACS. From the depleted fraction, CD71(+) erythroi d cells were enriched up to 80 per cent by MACS. This 'double-MACS' pr ocedure yielded an average depletion rate of 780-fold and an average e nrichment rate of 500-fold, with approximate recovery rates of 40-55 p er cent. For paternity testing, cells from unseparated blood and the v arious,fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45(-)/CD 71(+) sorted cells from maternal blood, but not in unfractionated cell s 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 we eks 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.