RARE EVENT SELECTION OF FETAL NUCLEATED ERYTHROCYTES IN MATERNAL BLOOD BY FLOW-CYTOMETRY

A noninvasive method of prenatal genetic diagnosis requires fetal cell selection from the maternal circulation that allows efficient recover y for analysis by fluorescence in situ hybridization (FISH), We have s olved several problems that negatively affect the isolation and FISH a nalysis of fetal nucleated red blood cells (nRBCs) in the maternal cir culation, The use of glycophorin A (Gly A) antibodies (Abs) for select ion is problematic because all five monoclonal antibodies (mAbs) teste d caused agglutination of non-nRBCs, thereby changing both light scatt er and fluorescence properties of cells by flow cytometry, Because the number of non-nRBCs is variable after Ficoll separation, isolation of nRBCs could be compromised severely by agglutination of nucleated cel ls with nonnucleated cells, causing them to shift light scatter and fl uorescence properties, Several methods for the removal of unwanted mat ernal white blood cells with CD45 mAbs were also evaluated, Magnetic b ead depletion was found to interfere with FISH detection because of re sidual bead debris after sorting, By contrast, removal of CD45(+) cell s by a panning technique eliminated this problem, Positive selection m ethods based on CD71, CD45, and LDS-751 staining and detection of feta l cells by gamma globin expression were also analyzed, Fetal cells wer e detected by FISH in 11 of 19 (CD71 selection) and in 13 of 15 (gamma selection) random pregnancies, These data support the possibility of a noninvasive method for isolation and analysis of fetal cells for pre natal diagnosis. (C) 1996 Wiley-Liss, Inc.