Enrichment, immunomorphological, and genetic characterization of fetal cells circulating in maternal blood

Abstract

Fetal cells circulating in the peripheral blood of pregnant women are a potential target for noninvasive genetic analyses. They include epithelial (trophoblastic) cells, which are larger than peripheral blood leukocytes. We enriched circulating trophoblastic cells using the isolation by size of epithelial tumor cells (ISET) method. Peripheral blood was obtained at 11 to 12 weeks of pregnancy. Cells isolated by ISET were stained by hematoxylin and eosin or by immunohistochemistry. Large epithelial cells were microdissected and fetal cell identification was obtained by polymerase chain reaction with short tandem repeats and/or Y-specific primers. By analyzing only 2 ml of blood, we found a variable number (n = 1 to 7) of Y-positive cells (overall 15 of 23) in all of the six mothers carrying a male fetus. In contrast, none of the 26 cells isolated from seven mothers carrying a female fetus scored positive. Eleven cells were analyzed by using short tandem repeat-specific markers: six of them showed a fetal profile and five showed a maternal profile consistently with Y-specific results. Only one-fifth of the single cell DNA was used for fetal cell assessment, leaving enough material for further genetic tests. We also show that the ISET approach allows the performance of fluorescence in situ hybridization analyses and the detection of DNA point mutations in single microdissected cells. We conclude that this is a powerful approach to enrich circulating fetal cells and prove their fetal origin, and that it may have implications for noninvasive prenatal diagnosis of genetic disorders.

Figures

Figure 1.

Top: Specificity test of Y primers (lanes 1 to 21) on PBL-derived DNA obtained from 20 women (lanes 1 to 12 and 14 to 21) and one man (lane 13, positive control). Bottom: Amplification of fetal Y-chromosomal sequences (198 bp) in single large cells isolated from maternal blood. Lanes 22 to 42: Single cells isolated from mothers carrying a male fetus (lanes 23, 25, 27, 29, 31, 33, 34, 36, and 38). Fetal Y-positive cells: lanes 25, 29, 33, 34, 36, and 38. Maternal Y-negative cells: lanes 23, 27, 31. Negative controls: buffer without sample inserted at the cell lysis step and run to the end of the test: lanes 22, 24, 26, 28, 30, 32, 35, 37, and 39. Positive controls: one single HuH6 cell (lane 40), 5 ng and 2 ng of male PBL-derived DNA (lanes 41 and 42). M, molecular weight marker (φX174 HaeIII digested).

Figure 2.

A to F: Morphological and immunohistochemical characterization of circulating cells isolated by ISET and proved to be fetal by Y-specific single-cell PCR testing A: Mononucleated cell (diameter, 27.5 μm) with cytotrophoblastic-like morphology laying on a filter pore (round mark in the nucleus). Three empty pores are visible on the top right and a neutrophil on the bottom left of the picture (H&E staining). B: Polynucleated, syncytiotrophoblastic cell (H&E). C: Cytotrophoblastic cell positive to the KL1 antibody. D: Syncytiotrophoblastic cell positive to the anti-placental alkaline phosphatase antibody. E and F: Maternal cells positive to the anti-leukocyte common antigen antibody. G: FISH analysis performed with a X-specific probe on HuH7 cells mixed with blood and filtered. The majority (∼98%) of the filtered cells are labeled. Two cells are shown in the inset at higher magnification (×60). H: Sequence analysis of β-catenin exon 3 showing the G to T mutation leading to the Gly to Val mutation (codon 34) in a single HuH6 cell. Original magnifications: ×10 (G); ×40 (B and D); ×60 (A, C, and E); ×80 (F).

Figure 3.

Genotyping by STR markers of parental and trophoblastic DNA and of single-cell DNA isolated from the maternal blood and microdissected. Electrophoretograms of the amplified products obtained using the STR marker D16S3018. A: The STR marker does not distinguish paternal (F) from maternal (M) alleles (A1 and A2: 249 and 261 bp). The trophoblastic DNA (T) shows homozygosity for one (249 bp) allele and the same pattern is found in two fetal cells (FC). B: The trophoblastic DNA (T) shows the maternal allele (MA: 256 bp), also found in the maternal DNA (M), and the paternal allele (FA: 258 bp). The same two alleles were detected in two microdissected fetal cells (FC).