Noninvasive prenatal diagnosis of fetal aneuploidy by circulating fetal nucleated red blood cells and extravillous trophoblasts using silicon-based nanostructured microfluidics

Circulating fnRBC and EVT captured by PicoBioChip

The scanning electron microscope (SEM) micrograph of the PicoBioChip is shown in Fig. 2a. The micrograph demonstrated that the PicoBioChip surface morphology is composed of patterned nanostructure with the same dimension and space, so that circulating cells could be captured by the chip surface (Fig. 2b). The processes of cells capture were automatically performed on a Cell Reveal™ system. By using a fluorescence microscope, the nRBC and EVT can be unequivocally distinguished from the background packed with the WBC from the maternal origin (Fig. 3).

Capture efficiency

Fluorescence in situ hybridization (FISH)

Interphase FISH for the captured fetal cells from the blood of pregnant women with a fetus of trisomy 13, trisomy 18, or trisomy 21 revealed correct diagnoses in all cases. The number of fnRBC and EVT examined ranged from one to ten for each case (Table 1). FISH for the trisomy 13 revealed nuc ish(RB1/D13S1195/D13S1155/D13S915x3, D21S270/D21S1867/D21S337/D21S1425/D21S1444/D21S341x2), for the trisomy 18 revealed nuc ish(D18Z1x3,DXZ1x2), and for the trisomy 21 revealed nuc ish(RB1/D13S1195/D13S1155/D13S915x2,D21S270/D21S1867/D21S337/D21S1425/D21S1444/D21S341x3) (Fig. 4).

Whole genome amplification (WGA)

All pooled captured cells underwent WGA successfully except those the total numbers of cells were too few (namely, less than 4 cells) to reach the amplified threshold for subsequent molecular genetic analyses by short tandem repeat (STR) analysis, aCGH, and NGS. Overall, fnRBC WGA from all the five cases and EVT WGA from two cases were obtained (Table 1). The WGA products were 50 μl in total with a concentration ranged from 290 to 844 ng/μl.

Short tandem repeat (STR) analysis

Array comparative genomic hybridization (aCGH) and next generation sequencing (NGS)

Both of aCGH and NGS analyses were performed for the captured fnRBC from the five cases and all the cases were correctly diagnosed. The results of aCGH are comparable with that of NGS (Fig. 5), and are consistent with the karyotyping results.

Samples

During 2016–2017, 24 women who carried the singleton pregnancy and received the first trimester serum screening for Down syndrome at GA 11–13 + 6 weeks or who decided to receive NIPT, was asked to donate blood sample to be used for validation. For each individual, 4 ml additional blood was stored in the BD vacutainer® with ACD solution A (Becton, Dickinson and Company, New Jersey, USA) for cbNIPD. When verification, at 2017 another five pregnant women carrying the singleton pregnancy at first or second trimester who decided to receive invasive procedures (chorionic villus sampling or amniocentesis) were recruited as a research basis to receive paralleled cfDNA testing (i.e. NIPT) and cbNIPD after informed consents (with the approved protocol CCH-IRB-141219) were signed. For each individual, approximately 20 ml of venous blood were collected. The blood was taken and stored in the Streck Cell-Free DNA BCT® (Streck, Nebraska, USA) for NIPT (12 ml) and in the BD vacutainer® with ACD solution A (Becton, Dickinson and Company, New Jersey, USA) for cbNIPD (8 ml). A total of 3 pregnant women who had singleton pregnancy affected with fetal aneuploidy were recruited, including trisomy 13 (n = 1), trisomy 18 (n = 1), and trisomy 21 (n = 1) fetuses respectively. Meanwhile, two women carrying the euploid fetuses (46,XX, n = 1 and 46,XY, n = 1) were also enrolled. The pregnant women were enrolled at first or second trimester ranged from gestational age 11⁺⁴ to 21 weeks (Table 1). It is noteworthy that the examiners of the cbNIPD lab have no prior knowledge of the karyotyping results, namely, they were blind to the results to avoid ascertainment bias. The recruitment of patients, collection of samples, and conduct of research projects, were approved by the Ethical Commitees of the medical institutions where the samples were collected (the Taiwan Adventist Hospital, Taipei, Taiwan, and the Changhua Christian Hospital, Changhua, Taiwan).

PicoBioChip manufacture

The PicoBioChip is a Si nanostructure with a porous morphology that is fabricated using the metal-assisted chemical etching (MACE) technology. The fabrication sequence is described as followed: the starting materials are p-type (100) silicon wafers which followed standard cleaning procedures to remove environmental contaminants. The pattern of the PicoBioChip is defined by standard photolithographic techniques. The Ag film is deposited onto the silicon wafer in a HF/AgNO₃ mixture solution, and the wafers are etched in a HF/H₂O₂mixture solution. Then, after the etching step and the Ag film removal, a Si nanostructure with a porous morphology is formed that is a “nano-on-nano” structure. To enhance the capturing effect, the potential targeted cells are pre-labeled with biotinylated antibodies and the PicoBioChip surface is made from a streptavidin material which has a specific binding interaction with biotin. The streptavidin-biotin is the strongest non-covalent biological interaction currently known. Via streptavidin-biotin interaction, biotinylated antibodies can be conjugated, enabling a high efficiency for targeted cells capture. The manufacturing flowchart, nano-on-nano structure and capture conception of the PicoBioChip are shown in Fig. 1.

Circulating fetal cells captured by cell reveal™ system with PicoBioChip

The whole blood sample (8 ml) is flown through the automated Cell Reveal™ system and then CFC are captured by PicoBioChips. For each run of test, four PicoBioChips were used: two for fnRBC capture and two for EVT capture. The antibodies used for primary capture of circulating fetal cells are CD71⁺ for fnRBC and EpCAM⁺ for EVT. PicoBioChips are examined using a fluorescence microscope equipped with a built-in automatic inspection and image analysis system, called the Cell Analysis Tool (CytoAurora CAT™), to filter out images of maternal white blood cells (WBC) for further analyses. The fnRBC and EVT can therefore be targeted, identified and enumerated. Image analyses with the count-in/filter-out criteria for different cell types are CD71(+)/GPA⁺(glycophorin-A)/CD45⁻/DAPI⁺ for fnRBC and CK7⁺(Cytokeratin-7)/HLA-G(+)/CD45⁻/DAPI⁺ for EVT, according to literatures and our in-house optimization [6, 21, 22, 35–40]. Namely, we first used one antibody to capture fnRBC and EVT separately, and then using other antibodies to stain the captured cells. Hence, the fnRBC were primarily captured by CD71 and then stained with CD71 and GPA, whereas the EVT were primarily captured by EpCAM and then stained with CK7 and HLA-G. Namely, we utilized dual antibodies (CD71 and GPA) to delineate the fnRBC and triple antibodies (EpCAM, CK7, HLA-G) to delineate EVT. It is noteworthy that in the validation group (n = 24) to validate the capture efficiency, only 4 ml maternal blood was used, in which 2 ml was for fnRBC (or more strictly, nRBC) and 2 ml was for EVT in each case. Only the five pregnant women enrolled for verification had 8 ml maternal blood to be withdrawn and used for cbNIPD.

Fluorescence in situ hybridization (FISH)

FISH was performed directly on one PicoBioChip capturing for fnRBC and one chip for EVT. Prior to hybridization, the formaldehyde on PicoBioChips were treated by 10 mM sodium citrate at 90 °C for 20 min, followed by being immersed in 0.1% Triton-X at room temperature for 10 min, then followed by serial washes of 0.2 N HCl at 25 °C for 20 min, purified water (double distilled) at 25 °C for 3 min and 2X SSC at 25 °C for 3 min, and an immersion of Vysis pretreatment solution (1 N NaSCN) (Abbott, IL, USA) at 25 °C overnight. Then, the PicoBioChips were deposited in purified water at 25 °C for 1 min, 2X SSC at 25 °C for 5 min (repeated two times), pepsin solution (10 μl 10% Pepsin / 40 ml 0.01 N HCl) at 37 °C for 3 min and 2X SSC at 25 °C for 5 min (repeated two times). Finally, the PicoBioChips were immersed in 70% ethanol at 4 °C for 1 min, 85% ethanol at 4 °C for 1 min and 100% ethanol at 4 °C for 1 min, and dried at 50 °C for 5 min. Interphase FISH for chromosome 13, 18 and 21 on captured fnRBC and EVT was then conducted using Aquarius® FAST FISH Prenatal kit (Cytocell, Cambridge, UK). For hybridization experiment, the PicoBioChips were dehydrated in an ethanol series and hybridized overnight in a moist chamber at 37 °C. The chips were washed for 2 min in 0.4X SSC at 70 °C and for 5 min in 4X SSC, 0.1% Tween 20 at room temperature and blocked in 4X SSC, 3% bovine serum albumin (BSA), 0.1% Tween 20 at 37 °C for 30 min. The hybridization signal was detected with Nikon-Ni-E microscope system (Nikon, Tokyo, Japan). Chromosomes were counterstained with 0.125 μg/ml DAPI in Antifade (Vysis, Illinois, USA). FISH analyses were performed using the Aquarius® FAST FISH Prenatal kit (Cytocell). The chromosome 13 probe for RB1, D13S1195, D13S1155, and D13S915, the chromosome 18 probe for centromere of chromosome 18(D18Z1), the chromosome 21 probe for D21S270, D21S1867, D21S337, D21S1425, D21S1444, and D21S341, and the chromosome X probe for centromere of chromosome X (DXZ1) were labeled with green, aqua, orange, and green fluorophores, respectively.

Retrieval of captured cells by PicoBioChip

The captured fnRBC and EVT are separately released by with capillary micropipette from PicoBioChips which are destined for DNA analyses. The location of captured cells-on-chip is acquired by the CytoAurora CAT™. Capillary micropipette crashes the chip’s nano structure of the target captured cells. The captured cells on the chip surface are followed by capillary micropipette picking up, which allows captured cells to escape from chip to be released for sequential analyses.

Whole genome amplification (WGA)

The captured fetal cells retrieved from the same PicoBioChip are pooled. The fnRBC and EVT were subjected separately to WGA, with 1.8 μg/μl BSA serving as the blocking agent to reduce the surface interaction from the silicon debris. WGA was performed using REPLI-g Single Cell Kit (Qiagen, Hilden, Germany) and following the manufacturer’s instructions. Amplified DNA was purified using the QIAamp DNA Blood Mini Kit (Qiagen). The DNA purities and concentrations were examined by Qubit fluorometer (Thermo Fisher Scientific, Delaware, USA) and Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific).

Short tandem repeat (STR) analysis

STR analysis was performed to confirm that the circulating cells captured and WGA DNA of fnRBC and EVT are indeed from fetuses instead of maternal origin. GenomeLab Human STR Primer Set kit (Beckman Coulter, California, USA) containing 12 primer pairs to amplify 11 STR loci and one gender-specific locus (Table 2) was used to analyze patterns of the STR by capillary electrophoresis according with the supplier’s protocol. PCR products were run on GenomeLab™ GeXP Genetic Analysis System (Beckman Coulter). FRAGMENTS application program (Beckman Coulter) was used for data collection and allele sizing.

Array comparative genomic hybridization (aCGH)

Approximately 1000 ng of WGA DNA was subjected to aCGH by GenetiSure Pre-Screen Array Kit 8x60K (Agilent Technologies, CA, USA), following the manufacturer’s instructions. The image on a chip was acquired with a G4900DA SureScan microarray scanner (Agilent Technologies, CA, USA) and analyzed with Agilent CytoGenomics software (Agilent Technologies) for chromosome gain or loss. Aberrations were detected by using default setting.

Next generation sequencing (NGS)

Approximately 1000 ng of WGA DNA was used for library construction using Ion Xpress Plus gDNA Fragment Library Preparation Kit Set (Thermo Fisher Scientific, California, USA) and following the manufacturer’s instructions. The quantity of library was determined using Qubit dsDNA HS assay kits (Thermo Fisher Scientific) with Qubit fluorometer (Thermo Fisher Scientific). The template-positive Ion Sphere Particles were generated using Ion PGM Hi-Q Template Kits (Thermo Fisher Scientific) with the Ion OneTouch 2 Instrument (Thermo Fisher Scientific) and then enriched with the Ion OneTouch ES Instrument (Thermo Fisher Scientific). Sequencing was performed on the Ion Torrent PGM Instrument (Thermo Fisher Scientific) platform using the Ion PGM Hi-Q Sequencing Kit and Ion 316 chip (Thermo Fisher Scientific). Analysis of the WGA product being sequenced was performed by using the cloud-based the Ion Reporter™ Server System (https://ionreporter.thermofisher.com/ir/).