Novel and High‐Throughput Method of Isolating Single Fetal Cells Using FACS for NIPT

Abstract:

Objective: To evaluate fluorescence activated cell sorting (FACS) as a method of single‐cell isolation of rare circulating fetal
cells from maternal blood for use in cell‐based non‐invasive prenatal testing (cbNIPT).
Method: Blood samples (30 mL) were collected from 75 ‘low‐risk’ pregnant women (gestational age 10–15 weeks). Fetal cells
were enriched and stained using magnetic activated cell sorting. Following enrichment, single fetal cells were sorted in individual
PCR tubes by FACS. After cell lysis, verification of fetal cell origin was performed using short tandem repeat (STR)
analysis with the GlobalFiler PCR Amplification kit.
Results: An average of 13.7 cells were sorted using FACS. STR analysis identified 8.2 fetal cells on average, representing 60.2%
of the sorted cells. The four‐step single‐cell isolation procedure facilitated an overall enrichment of approximately 16‐millionfold.
One sample did not render any fetal cell, corresponding to 1.3% of the samples.
Conclusion: FACS, which is typically used for segregation of large populations of cells, can be used for single‐cell isolation of
rare fetal cells in an automated setup. This not only helps in making cell isolation faster and high throughput but also provides
fetal cells for a more comprehensive genetic analysis of the fetus.Introduction: Cellular derangements contributing to fetal membrane damage precede spontaneous rupture at term and are even more pronounced in preterm prelabor rupture of membranes. We hypothesized that such pathophysiological processes lead to shedding of fetal membrane-derived cells into maternal blood. This study aimed to identify fetal membrane cells in maternal blood and to examine whether their concentration correlates with membrane rupture.

Methods: Blood samples (30 mL) were collected from pregnant women. Using antibodies targeting proteins highly expressed in the fetal membranes, fetal cells were enriched by magnetic activated cell sorting and isolated by fluorescence activated cell sorting. Their fetal origin was confirmed by short tandem repeat analysis.

Results: Fetal cells were detected in 28% of samples (22 of 78). Detection rates and concentrations varied across groups: (1) spontaneous or iatrogenic term rupture of membranes (≥37 weeks), 50% (10/20), 1-12 cells; (2) term not in labor (≥37 weeks), 27% (4/15), 1 cell; (3) preterm prelabor rupture of membranes (<34 weeks), 17% (3/18), 1 cell; (4) preterm labor with intact membranes (<34 weeks), 10% (1/10), 1 cell; and (5) preterm not in labor (<34 weeks), 27% (4/15), 1-3 cells. After rupture of membranes at term (group 1), the detection rate decreased with time, and no cells were found more than 10 h post-rupture.

Discussion: The protocol successfully isolates fetal cells from maternal blood, potentially originating from the fetal
membranes. Their concentration was highest after term rupture of membranes but does not appear to increase
after preterm rupture compared with other pregnancies.