Evidence for feasibility of fetal trophoblastic cell-based noninvasive prenatal testing

Key findings

• Fetal cells were recovered from most samples and confirmed as fetal in origin by Y-chromosome PCR or short-tandem-repeat genotyping, including more than 30 genotypically male cells plus individual cells carrying trisomy 13, trisomy 18, trisomy 21, and a 47,XXY karyotype.
• Genome-wide analysis reached subchromosomal resolution: a 2.7 Mb deletion on chromosome 15q was detected from two individual fetal cells, below the reach of current cell-free NIPT, while a smaller 1.2 Mb deletion went undetected in four cells.
• Fetal-cell recovery averaged 0.74 cells per milliliter of maternal blood, falling to 0.36 per milliliter once a white-blood-cell depletion step was added to raise sample throughput.

The AccuCyte–CyteFinder workflow in the methods

“Fetal cell enrichment was carried out with RareCyte's trophoblast enrichment and staining method. This has been described for the identification and retrieval of circulating tumor cells (CTCs) 21 and was modified to allow for retrieval of rare target cells from liquid slide preps. Briefly, 6 mL of preserved blood was added to the AccuCyte® Separation (ACS) tube (RareCyte, Inc., Supporting Information Figure S1A ). 21 In an attempt to reduce the number of slides to be scanned in order to increase the throughput of the test, a step for WBC depletion was explored. In the majority of samples where WBC depletion was performed, RosetteSep Human CD45 and CD36 Depletion Cocktails (STEMCELL Technologies) were combined with the blood for 20 min at room temperature (RT). In some cases a slightly modified WBC depletion protocol was carried out but both gave equivalent WBC depletion and fetal cell recovery results. A float (density 1.058–1.061 g/cm 3 ) was then added to the ACS tube. The sample was centrifuged at 5250 × g for 30 min, a sealing ring was applied to the outside of the ACS tube below the WBC band on the float, and the plasma was aspirated. Next 150 μL of a high‐density retrieval fluid (Fluid A) was added, and the ACS tube was centrifuged at 1000 × g for 5 min displacing the less dense WBCs to above the float. A second sealing ring was placed at the top of the float to keep the WBCs in solution above the float. Then 30 μL of 5% paraformaldehyde (Ted Pella, Inc.,) in PBS was added. After 20 min, cells were stained for 1 h at RT by adding a blocking/permeabilizing solution, followed by staining cocktail containing DAPI (BioLegend), anti‐cytokeratin Alexa‐488 (eBiosciences, clone AE1/AE3), anti‐cytokeratin Alexa‐488 (BioLegend clone C11), and anti‐CD45 PE (BioLegend clone 2D1). Fetal cell recovery Following staining, the cells were collected and removed from the antibody cocktail as follows. Briefly, 1 mL of a density increasing reagent (Fluid A) was mixed into the staining solution slurry. The EpiCollector® (RareCyte, Inc.) device was then inserted into the ACS tube and a second fluid of lesser density (Fluid B) was gently layered on top of the denser staining solution slurry. A 1.5‐mL isolation tube containing 200 μL of a 1.1 g/cm 3 collection fluid (Fluid C), less dense than Fluid B, was inserted into the EpiCollector. The ACS tube, now containing a gradient of density fluids without air gaps, was centrifuged at 1000 × g for 20 min. During centrifugation, cells that have density < 1.1 g/cm 3 float upward out of the denser fluids into Fluid C within the collection tube. The cells have thus been drawn through the antibody solution, removing unbound antibody in a ‘pseudo‐wash’ process. The isolation tube was then removed from the EpiCollector, 800 μL of PBS was added and stored at 4 °C until imaging. Automated image capture and analysis Stained cells were pipetted into custom well slides (CyteSlide, RareCyte, Supporting Information Figure S1B ) that were placed onto the CyteFinder digital scanning microscope to acquire fluorescent images ( Supporting Information Figure S1C, S2 ). The CyteFinder acquired 4‐channel fluorescent images of low magnification (10 × objective) fields of view for each CyteSlide covering the entire well.”

— Breman et al., Prenatal Diagnosis (2016), Materials and Methods, “Fetal cell enrichment and staining”

Disclosure: RareCyte is listed as an author affiliation on the publication cited above.

Disclosure: RareCyte is named in the competing-interests statement of the publication cited above.

Why it matters for The AccuCyte–CyteFinder workflow users

If your work turns on finding a few rare cells in a large background and lifting them out intact, this study puts the AccuCyte–CyteFinder workflow through that test in a demanding new setting. The team drew maternal blood, used the AccuCyte enrichment step to separate and stain nucleated cells by density, then scanned each slide on the CyteFinder to flag the cytokeratin-positive, CD45-negative trophoblasts hiding among maternal cells and retrieve them one at a time. The rare-cell target here is fetal rather than a tumor cell, but the demand on the instrument is the one you already know: locate a scarce, specifically stained cell and recover it without dragging along its neighbors. What makes the result count is what happened next, because the single cells they picked yielded enough DNA for whole-genome amplification, array CGH, and sequencing, including a copy-number change just 2.7 Mb across. For your own rare-cell problem, the lesson is that an enrichment-and-retrieval workflow proven on circulating tumor cells extends to any rare cell you can stain and image.