Detection of chromosomal aneuploidies in fetal cells isolated from maternal blood using single‐chromosome dual‐probe FISH analysis

Abstract

Detection of chromosomal aneuploidies using fetal cells isolated from maternal blood, for prenatal non-invasive genetic investigation, has been a long-sought goal of clinical genetics to replace amniocentesis and chorionic villous sampling to avoid any risk to the fetus. The purpose of this study was to develop a sensitive and specific new assay for diagnosing aneuploidy with circulating fetal cells isolated from maternal blood as previously reported using two novel approaches: (i) simultaneous immunocytochemistry (ICC) evaluation using a monoclonal antibody for i-antigen, followed by fluorescence in situ hybridization (FISH); (ii) dual-probe FISH analysis of interphase nuclei using two differently labeled probes, specific for different loci of chromosomes 21 and 18; in addition, short tandem repeats (STR) analysis on single cells isolated by micromanipulation was applied to confirm the presence of fetal cells in the cell sample enriched from maternal blood. Blood samples were obtained from women carrying trisomic fetuses, and from non-pregnant women and men as controls. Using ICC-FISH approach, a large heterogeneity in immunostaining pattern was observed, which is a source of very subjective signal interpretation. Differently, dual-probe FISH analysis provided for a correct diagnosis of all pregnancies: the mean percentage of trisomic cells was 0.5% (range, 0.36-0.76%), while the mean percentage of trisomic cells in the control group (normal pregnancies or non-pregnant women) was ≤0.20%. The application of the dual-probe FISH protocol on fetal cells isolated from maternal blood enables accurate molecular detection of fetal aneuploidy, thus providing a foundation for development of non-invasive prenatal diagnostic testing.

Characterization of Fetal Cells from the Maternal Circulation by Microarray Gene Expression Analysis - Could the Extravillous Trophoblasts Be a Target for Future Cell-Based Non-Invasive Prenatal Diagnosis?

“…A major disadvantage of both of these procedures is, however, that the sampling procedure is associated with a risk of unintended pregnancy loss in the range of 0.5-1% [1]. Therefore, researchers have for a long time tried to use maternal blood samples for non-invasive prenatal diagnosis (NIPD) since it is envisioned that fetal cells isolated from the maternal circulation can replace cells from amnionic fluid or chorionic villi in diagnosis for common chromosomal aneuploidies using fluorescence in situ hybridization (FISH)-based techniques [2]. However, fetal cells in the maternal circulation are extremely rare with estimates of 1-2 cells/ml blood [3,4].…”

Aneuploidy screening using circulating fetal cells in maternal blood by dual‐probe FISH protocol: a prospective feasibility study on a series of 172 pregnant women

“…All other patterns of hybridization were excluded from the analysis, although they were recorded for hybridization quality control (Calabrese et al. ). Multinuclear cells, representing exceptional findings, were excluded in the FISH scoring process to avoid FISH signals misinterpretation.…”

“…Fluorescence in situ hybridization investigation was performed according to the procedure previously described dual-probe FISH analysis; ref. Calabrese et al 2012). Using the protocol described by Yan et al (2000) which removes the cytoplasm and swells nuclei thus facilitating FISH analysis, cells from enriched samples were incubated in prewarmed (37°C) hypotonic KCl (0.075 mol/L) for 5 min and fixed three times with cold Carnoy's fixative (methanol:glacial acetic acid, 3:1) before cytocentrifugation on glass slides.…”

“…A fluorescence in situ hybridization (FISH) analysis of these fetal cell‐enriched samples was carried out using two independent probes for chromosomes 18 and 21 (Calabrese et al. ). A correct diagnosis was achieved for all pregnancies; the mean percentage of trisomic cells was 0.5% (range 0.36–0.76%) in the aneuploidy group compared with ≤0.20% in the control group (normal pregnancies or nonpregnant women).…”

“…We previously described a method for enriching fetal cells from maternal blood, including epsilon-Hb-positive nucleated red blood cells (NRBCs), i-positive, and CD34positive cells (Sitar et al 2005). A fluorescence in situ hybridization (FISH) analysis of these fetal cell-enriched samples was carried out using two independent probes for chromosomes 18 and 21 (Calabrese et al 2012). A correct diagnosis was achieved for all pregnancies; the mean percentage of trisomic cells was 0.5% (range 0.36-0.76%) in the aneuploidy group compared with ≤0.20% in the control group (normal pregnancies or nonpregnant women).…”