Studies on the Isolation and Identification of Fetal Nucleated Red Blood Cells in the Circulation of Pregnant Women before and after Chorion Villus Sampling

Abstract

Objective: To investigate the feasibility of various molecular forms of hemoglobin as markers for fetal nucleated red blood cells (NRBCs). Methods: The presence of epsilon and gamma globin positive NRBCs was investigated in pure fetal blood and in blood from pregnant women before and after chorion biopsy. Maternal samples were enriched for NRBCs by various conventional methods, including limited enrichment by only positive CD71 selection or single density gradient. We searched for fetal cells on slides by automated scanning. Fetal cells were defined by (1) the presence of epsilon or gamma globin and (2) simultaneously by the presence of a Y chromosome signal. Results: 18 of 25 gamma globin positive cells identified in blood samples after chorion biopsy were chromosome Y signal positive, and 1 cell had two X chromosome signals. 263 of 339 epsilon globin positive cells identified in blood samples after chorion biopsy were hybridized with X and Y chromosome probes. None had two X signals, and 249 were Y positive. In blood samples before chorion biopsy, only 1 epsilon globin positive fetal NRBC and no epsilon globin positive maternal NRBCs were found. Conclusions: Epsilon globin may be specific for fetal NRBCs. Only 1 epsilon globin positive fetal cell was identified in 1 of 12 blood samples before chorion biopsy, representing a total of 182 ml of maternal blood. This suggests that most fetal cells found in maternal blood by fluorescence in situ hybridization methods may not be NRBCs.

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?

“…Cells on slides were fixed, permeabilized and X and Y chromosome-specific FISH was performed as described [11] with omission of the rehybridization step. Automated microscope scanning for finding cells with a Y chromosome was also performed as described previously [11].…”

“…Automated microscope scanning for finding cells with a Y chromosome was also performed as described previously [11]. …”

“…However, fetal cells in the maternal circulation are extremely rare with estimates of 1-2 cells/ml blood [3,4]. For this reason several research groups have tried to establish technologies for the enrichment and identification of a range of fetal cell types, including nucleated red blood cells, certain types of fetal white blood cells and placenta-derived cells but with limited success [5,6,7,8,9]. One possible reason for this lack of success is limited knowledge on the specific types of the fetal cells present in maternal blood, leading to suboptimal markers for enrichment and identification.…”

New trends in non-invasive prenatal diagnosis: Applications of dielectrophoresis-based Lab-on-a-chip platforms to the identification and manipulation of rare cells (Review)

“…The isolation of rare cells from maternal blood for noninvasive prenatal diagnosis is a new field of research exhibiting several difficulties since this strategy requires several basic technological protocols for a successful outcome (19,45,46,48,50,52,62,68,(104)(105)(106)(107)(108)(109)(110)(111)(112).…”

The Number of Endovascular Trophoblasts in Maternal Blood Increases Overnight and after Physical Activity: An Experimental Study

“…Whereas noninvasive prenatal testing (NIPT) based on cell-free fetal DNA is currently being implemented clinically worldwide, numerous attempts to establish NIPD based on fetal cells in maternal blood have failed [1,2,3,4,5]. This is primarily due to the very low number of fetal cells in maternal blood [2].…”