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 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.
Büsch
J
J
Institute for Forensic Medicine, University of Bonn, Germany.
Huber
P
P
Pflüger
E
E
Miltenyi
S
S
Holtz
J
J
Radbruch
A
A
D015703:Antigens, CD / Q000032:analysis; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D001483:Base Sequence; D002469:Cell Separation / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D005260:Female; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005838:Genotype; D006683:HLA-DQ Antigens / Q000235:genetics; D059848:HLA-DQ alpha-Chains; D006801:Humans; D016130:Immunophenotyping; D017493:Leukocyte Common Antigens / Q000032:analysis; D018950:Lipopolysaccharide Receptors; D008280:Magnetics; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011247:Pregnancy; D011990:Receptors, Transferrin
D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D015214:Antigens, Differentiation, Myelomonocytic; C068126:CD71 antigen; D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; C069050:HLA-DQA1 antigen; D018950:Lipopolysaccharide Receptors; D011990:Receptors, Transferrin; D004247:DNA; D017493:Leukocyte Common Antigens