Fetal Cell Isolation Market Overview
Scientists have been aware of the presence of fetal cells in maternal blood for over a century. These fetal cells are rare with one to two fetal cells per milliliter of maternal blood. This rarity, combined with the lack of knowledge of fetal cell specific markers, has until farily recently made the enrichment and detection of fetal cells for prenatal genetic testing quite difficult. Fetal cell isolation from maternal blood for non-invasive prenatal testing is done either via methods which analyze cell-free fetal (cff) DNA (fragments of dead fetal cells) or via methods which analyze circulating fetal cells (CFCs). Some current techniques to isolate fetal cells from maternal blood are as follows: cffDNA analysis using centrifugal techniques, cffDNA isolation using extraction kits, droplet digital PCR (ddPCR), DNS with STR process to separate CFCs, CFC and EVT capture using the Cell Reveal™ platform. Meanwhile, methods currently in development or testing include cell-based noninvasive prenatal diagnosis (cbNIPD) application on CFCs and the use of antibody-drenched chip for fetal cell isolation. These techniques are discussed in greater detail below.
cell-free fetal DNA (cffDNA) techniques
- Fluorescence-Activated Cell Sorting
The basic method of isolating fetal cells involves "fluorescence-activated cell sorting". This approach utilizes light to separate mixed specimen cells. However, this involves sorting through the specimen cell by cell.
- Cell Separation Using Compounds
Traditional strategies of isolating fetal cells involve the use of conservative compounds such as formaldehyde to make the separated maternal white blood cells (wbc) to be more stable. This makes the isolated blood cells to resist cell wall lysis disintegration.
- cffDNA Analysis Using Centrifugal Techniques
"Initial high speed and double centrifugation of serum" can also remove maternal white blood cells within the DNA isolation process. Instruments such as the SpeedVac Concentrator can perform this process. The concentrated maternal cffDNA can then be analyzed by instruments such as the "Agilent Bioanalyzer 2100 instrument and Agilent DNA High Sensitivity Kit". The "Agilent 2100 Expert software" can then evaluate the DNA profile of each sample. Proprietary software can then show the electropherogram pattern for the specimen. However, the singled out cffDNA might be miniscule and of low quality.
- cffDNA Isolation Using Extraction Kits
cffDNA can be further isolated by using "QIAamp DNA Blood Mini Kit and AccuPrepTM Genomic DNA Extraction Kit." The resulting mixture can be further dissolved in ddH2O.
- Droplet Digital PCR (ddPCR)
Plasma quantification can also be performed by a "ddPCR assay" which is formulated to expand a short segment which is around 140 bp of the human RASSF1A gene promoter area. "Forward primer 5′- AGT GCG CGC GTG AGT AGT -3 and reverse primer 5- GGC GAA AGT AAC GGA CCT AGT-3" were built using "Roche ProbeFinder online software".
- cffDNA in Maternal Plasma
The "promotor region of the RASSF1A gene is hypermethylated in fetal DNA and hypomethylated in maternal DNA." This methylation gradient can be used to identify fetal DNA from maternal DNA. "Methylation-sensitive restriction enzymes" cannot process methylated areas of the genetic form. Hence, coating cffDNA from the blood plasma of pregnant women with "methylation-sensitive restriction enzyme BstUI" would result in the digestion of the hypomethylated promotor region of the maternal RASSF1A gene. This will leave the hypermethylated promotor area of the fetal RASSF1A gene to stay in form. These "undigested promotor regions of fetal RASSF1A gene can be quantified PCR amplification".
- Next Generation Sequencing
The cfDNA-based testing process is also used to identify the minor differences between maternal blood that has euploid fetuses and those blood cells that possess aneuploidy fetuses. This testing process is done via the "robust maximal parallel sequencing methods by utility of the high-sensitive, high-throughput, rapid-evolving platforms called next generation sequencing (NGS) technologies".
Under the microchip category, several processes have been tried before to isolate cells which include the following: "immune-affinity, immune-magnetic, and size-based techniques." Microchip-based appproach is done using immune-affinity strategy that uses a microfluidic device called "PicoBioChip." The device is layered with antibodies to capture the identified antigens on the targeted cells.
Circulating Fetal Cells (cfc) techniques
- DNS with STR to Separate CFCs
To isolate fetal cells, researchers have designed a "double negative selection (DNS) procedure that can indiscriminately enrich the fetal cells. After "short tandem repeat (STR)" validation, the separated circulating fetal cells (CFCs) were made to undergo an immersive "whole genome sequencing analysis".
- CFC and EVT Capture
"Circulating fetal nucleated red blood cells (fnRBC) and extravillous cytotrophoblasts (EVT)" can be captured using "silicon-based nanostructured microfluidics platform named as “Cell Reveal™.” The platform uses immunoaffinity to catch the "trophoblasts and the nucleated RBC (nRBC) with specific antibodies." The automated computer software is then used to analyze and isolate the targeted particles by applying more immunostaining to the antigens.
- Magnetic Bead Enrichment
Protocols for magnetic bead enrichment can also be enhanced to help in capturing fetal cells. The cells can be stained and certain algorithms can be configured in fluorescence microscopes to further pinpoint fetal cells amidst the sea of maternal cells. The fetal cells can then be transferred and further be processed in a localized microwell plate or tube.
- Magnetic Cell Sorting
Denmark's Arcedi Biotech employs a "cell-based noninvasive prenatal diagnosis (cbNIPD) testing" that utilizes cells that are already in maternal blood. The test is done on a blood specimen from a woman during the end of the first trimester of her pregnancy. The team dissects the red blood cells from the sample and keeps the leftover white blood cells. White blood cells are then combined with proprietary antibody solution on a magnetic cell sorting plate. Another set of antibody solutions that seeks out certain fetal cells are then introduced to stain the target cells. The fetal cells can then be pinpointed by magnifying them and using microarray processing.
- Next Generation Sequencing
Fetal trophoblasts have been extracted from around 30mL of maternal blood by applying the following processes: "maternal white blood cell depletion, density-based cell separation, immunofluorescence staining, and high-resolution scanning". Fetal cells were identified as single cells. These then went through a whole genome amplification process for "subsequent genome-wide copy number analysis and genotyping" to verify where the cell's origination.
TECHNIQUES UNDER DEVELOPMENT
- (cbNIPD) Techniques for Circulating Cells
New cell-based noninvasive prenatal diagnosis (cbNIPD) technologies might be further developed in the future to isolate both the circulating fetal cells (CFC) via technologies used to isolate other cells such as circulating tumor cells (CTC). These technologies include "PCR-based, flow cytometry, laser scanning cytometry, FDA-cleared Cell Search (Veridex, New Jersey, USA), EPISPOT assay, and microchip (microfluidics or lab-on-a-chip)-based technologies". The cbNIPD on EVT method can be further explored as a means to identify "de novo copy number variations".
- Antibody-Drenched Chip
An antibody-drenched chip that can capture fetal cells from maternal blood specimen is now being developed by researchers. They will then mark the trophoblasts with flourescent antibodies. Using "laser capture microdissection" method, they will then harvest only those cells that appear phosphorescent. This solution is expected to enable accurate genetic disorder tests through a routine blood extraction. The chip technology is in the process of being commercialized by FetoLumina Technologies.
- Microfluidic Device
A miniscule microfluidic device that can isolate fetal cells from maternal cells is currently under development to identify genetic mutations in early stage pregnancy via non-invasive blood tests. This small device was inspired by the design of a similar gadget that can isolate cancer cells from patients' blood. The tailored structure of the device can let huge volume of blood to be analyzed. This will then make the fetal cell separation process cheaper and speedier. The larger fetal cells can be easily separated by the device from the smaller maternal white blood cells. Researchers are now working with industry resources to make the device available for routine prenatal checkups.