Rare event selection of fetal nucleated erythrocytes in maternal blood by flow cytometry

Abstract

A noninvasive method of prenatal genetic diagnosis requires fetal cell selection from the maternal circulation that allows efficient recovery for analysis by fluorescence in situ hybridization (FISH). We have solved several problems that negatively affect the isolation and FISH analysis of fetal nucleated red blood cells (nRBCs) in the maternal circulation. The use of glycophorin A (Gly A) antibodies (Abs) for selection is problematic because all five monoclonal antibodies (mAbs) tested caused agglutination of non-nRBCs, thereby changing both light scatter and fluorescence properties of cells by now cytometry. Because the number of non-nRBCs is variable after Ficoll separation, isolation of nRBCs could be compromised severely by agglutination of nucleated cells with nonnucleated cells, causing them to shift light scatter and fluorescence properties. Several methods for the removal of unwanted maternal white blood cells with CD45 mAbs were also evaluated. Magnetic bead depletion was found to interfere with FISH detection because of residual bead debris after sorting. By contrast, removal of CD45+ cells by a panning technique eliminated this problem. Positive selection methods based on CD71. CD45. and LDS-751 staining and detection of fetal cells by gamma globin expression were also analyzed. Fetal cells were detected by FISH in 11 of 19 (CD71 selection) and in 13 of 15 (gamma selection) tandem pregnancies, These data support the possibility of a noninvasive method for isolation and analysis of fetal cells for prenatal diagnosis.