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 flow 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) random pregnancies. These data support the possibility of a noninvasive method for isolation and analysis of fetal cells for prenatal diagnosis. © 1996 Wiley‐Liss, Inc.

A comparison of different density gradients and antibodies for enrichment of fetal erythroblasts by MACS

“…Although the enrichment of fetal cells in our experiments described here was highest when using GPA, a characteristic cell clumping was observed under the microscopy light. A similar observation has previously been reported by Lewis et al (1996). Even though this aggregation of cells could cause a problem for FISH analysis, it should be no problem or could even be an advantage when picking cells for single-cell PCR analysis of genetic disorders (Garvin et al, 1998).…”

Quantification of all fetal nucleated cells in maternal blood in different cases of aneuploidies

“…We have observed a relatively high number of FNCs in all pregnancies with a trisomy 18 fetus. However, the total number of FNCs was statistically not higher (p ¼ 0.1570, Mann-Whitney Rank test) for pregnancies with 47,XY,þ18 (median 7; range [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] than that observed in normal male pregnancies as reported in our previous study (median 4; range 2-6) (1). Moreover, we compared the total number of FNCs observed in trisomy 18 vs the ones observed in trisomy 21 (data obtained from 16 women carrying male trisomic 21) (26).…”

“…Moreover, we compared the total number of FNCs observed in trisomy 18 vs the ones observed in trisomy 21 (data obtained from 16 women carrying male trisomic 21) (26). The total number of FNCs was not significantly higher (p ¼ 0.1568, Mann-Whitney Rank test) in the unit volume of maternal blood of women carrying male trisomic 21 fetus (median 10; range 6-32) than in the same volume of blood of women bearing fetus with trisomy 18 (median 7; range [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20].…”

“…The methods used to enrich maternal blood for fetal nucleated cells (FNCs) include FACS (10,(11)(12)(13)(14)(15), magnetic bead-activated cell sorting (MACS) (16)(17)(18)(19), and gradient density fractionation (17). These technologies may require specific equipment and expertise that are only available in selected referral centers.…”

Detection of gamma‐globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization

“…Routine isolation of fetal cells using a single antigen has not been successful owing to the lack of highly specific fetal markers. Some groups have investigated sorting of fetal cells by flow cytometry using antibodies against the gamma-globin chain of fetal hemoglobin (anti-HbF) in combination with DNA binding dyes (DeMaria et al, 1996;Lewis et al, 1996;Davis et al, 1998) and also antibodies against CD antigens for negative selection (de Graaf et al, 1999). However, the magnetic activated cell sorting (MACS) technique using antigen binding magnetic beads still seems to be the easiest way to enrich for NRBCs based on cell surface markers due to the large total number of cells (Hohmann et al, 2001).…”