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".
"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. 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.
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".
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.
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. Five companies have been identified who are actively working within the field of fetal cell isolation, specifically with circulating fetal cells as opposed to cell free DNA testing options. ARCEDI, Rarecyte, ANGLE, Racecells Diagnostics, and Abnova are active in publications, presentations, and testing around the globe. Each of the companies have been outlined below with a description of the technologies they are developing.
Based in Denmark, ARCEDI (an acronym for 'Advanced Rare Cell Diagnostics') Biotech is heavily invested in the development of new techniques and technologies surrounding cell-based noninvasive prenatal diagnosis (cbNIPD) and testing (cbNIPT). The company, in part with researchers Lotte Hatt, Palle Schelde Ripudaman Singh, has developed a proprietary method to 'enrich fetal cells from circulating maternal blood with high efficiency'. The extracted fetal cells are enriched for further analysis using more standard diagnostic technologies like FISH, array CGH, and Next Generation Sequencing which can detect chromosomal and sub-chromosomal changes within the fetal genome. As a pioneer in this field, ARCEDI is uniquely positioned to discuss both the pros and cons with the new advents within the cbNIPT technologies. A November 2018 paper released in part by the company and its researchers discussed challenges of isolating these rare fetal cells and then using them for prenatal genetic analysis. In this article, the researchers discuss the inherent challenges in developing a technology protocol which can isolate fetal cells because of the relatively 'few fetal cell types that are known to circulate in maternal blood'. Fetal cells are rare, with only one to two fetal cells per milliliter of maternal blood and thus the fetal cell testing technology developed needed to address the difficulty in both recovery and standards of purity within the samples retrieved. ARCEDI's technology finds this balance 'by optimizing protocols for magnetic bead enrichment followed by staining of the fetal cells, and also by developing algorithms for fluorescence microscopes for the identification of fetal cells in the background of maternal cells'. ARCEDI's mission is to 'provide a simpler, safer and accurate non-invasive prenatal diagnostic test to the pregnant women by analyzing the genome of the fetus using circulating fetal cells in maternal blood'. The company is currently scheduled to present at the Advanced in Prenatal Molecular Diagnostics Symposium in Lisbon, Portugal in May 2019.
RareCyte is located in Seattle, Washington, and is 'an innovative life science company that has developed technologies for the analysis of rare single cells in blood'. The company employs visions from both engineers and scientists in order to develop rare cell analysis technologies which 'include consumables, instrumentation, analysis software, and cell retrieval capabilities that enable advances in oncology, immuno-oncology, maternal-fetal health, and more'. Among the company's developments is an application in which 'circulating fetal cells (CFCs) can be isolated from maternal blood'. The RareCyte application is process which allows the 'enumeration of CFCs in maternal blood' and the 'retrieval of individual CFCs for molecular analysis by comparative genomic hybridization and next generation sequencing'. According to the company website, the key to the RareCyte system is the 'unbiased and highly sensitive target-cell recovery with the AccuCyte® Sample Preparation System'. This method allows for the 'capture and staining antibodies for isolation and detection of fetal cells, and visual confirmation protects against false positives' and for 'gentle, single cell isolation for detection of sub-chromosomal duplications and deletions'. Scientists from RareCyte co-authored an article titled 'Reliable Detection of Subchromosomal Deletions and Duplications Using Cell-based Noninvasive Prenatal Testing' in December 2018. This article discusses the methods employed in order to reach the conclusion that this 'cell-based NIPT method has the capacity to reliably diagnose fetal chromosomal abnormalities down to 1 to 2 Mb in size'. RareCyte will be represented at the Molecular Diagnostics Europe 2019 conference in May 2019 in Lisbon, Portugal and at the 2019 ASCO Annual Meeting in Chicago, Illinois in June 2019.
ANGLE has corporate presences in three countries, the United States, the United Kingdom, and Canada. ANGLE is a pioneer in the development of biopsy detection methods which use circulating cancer cells in order to diagnose diseases like breast and prostate cancers with solid tumor types. The company's Parsortix™ system is expected to be widely adopted by the global cancer treatment community as a diagnostic tool but is also in early FDA approval process for use in 'non-invasive fetal diagnostics, which involves harvesting fetal cells from the pregnant mother and analyzing for Down’s Syndrome and many other chromosomal and genetic conditions through a simple blood test'. In October 2018, ANGLE's founder and CEO, Andrew Newland, commented on the company's progress within the field of maternal testing at the 6th Annual Advances in Prenatal Diagnostics conference in Cambridge, Massachusetts. Mr. Newland's comments included this description of ANGLE's positioning within the market, 'non-invasive prenatal testing is a major new market for the Parsortix™ system and a potential solution to the current limitations of NIPT, providing a more comprehensive diagnosis of fetal abnormalities for mothers at risk of an affected pregnancy. ANGLE’s product-based approach to liquid biopsy enables us to leverage partnerships with third parties, which we are developing in the NIPT space, an established and fast growing large scale market'. Traditional NIPT, while becoming a preferred method for prenatal testing because of its relatively safety for both mother and fetus, has two chief limitations. The first of these limitations is that cell-free fetal (cff) DNA (fragments of dead fetal cells) is 'limited to testing for chromosomal disorders that are caused by the presence of an extra or missing copy (aneuploidy) of a chromosome'. The second limitation is the inherent barrier to companies entering the space because 'current methods are based on next generation sequencing analysis of cell-free DNA and the intellectual property is tightly held by a few large corporates'. Therefore, developing a method of NIPT that does not test cffDNA. The company's Parsortix™ system will allow for testing of intact fetal cells which will open 'the potential for whole genome analysis of the cell providing complete information on numerous genetic conditions and not just limited to a very small number of conditions'. ANGLE conducted a pilot study in which the Parsortix™ system was used to 'harvest and then analyze fetal cells from the blood of pregnant women'. The results of the study concluded that the system could be used to determine both XY chromosomes (in women expected to have a male child) and the trisomy 21 gene (in the blood of a mother whose fetus had been diagnosed with Down's Syndrome). Though a small-scale test, the company acknowledges 'substantial further work will be necessary to develop an effective NIPT test' and it hopes to improve upon the approximate 10% success rate of current cffDNA tests.
Founded by Patrizia Paterlini-Bréchot, an oncologist and hematologist and a professor of cellular and molecular biology at the University Paris Descartes, RareCells Diagnostics is based in Paris, France with a holdings subsidiary located in Harrison, New York. Prof. Paterlini-Bréchot has studied cancer cells since 1982 and in 1995 began focusing particularly on circulating cancer cells which led to the invention of ISET® technology in 2000. ISET® was the 'first, and still unparalleled, filtration method to isolate from blood, and without any bias, very rare Circulating Tumor Cells (CTCs), allowing their diagnostic identification by cytopathology and their molecular characterization'. This technology is now the base platform on which new and noninvasive prenatal diagnostics tests can be developed. As such, 'an ISET-like system has been developed for the isolation of Circulating Fetal Cells (CFC) from maternal blood (ISET-PND) to replace amniocentesis by a non-invasive genetic test for Trisomy-21, Cystic Fibrosis and other genetic disorder (ISET-PND test). Professor Paterlini-Bréchotinitially founded a company called Metagenex which initially held the ISET patents and licenses. The company fell victim to serious management issues and Professor Paterlini-Brechot disengaged herself from the company, retrieved the patents and licenses from the French public institutions and founded RareCells in 2010. Prof. Paterlini-Bréchot was a featured presenter at the ISPD 22nd International Conference on Prenatal Diagnosis and Therapy in Antwerp, Belgium in July 2018.
Abnova is the world's leading antibody manufacturer. The company, based in Taipei City, Taiwan, is capable of 'generating 300 mouse monoclonal antibodies and 200 rabbit polyclonal antibodies per month'. Most human expressed genes have been identified, after the human genome project, and full-length cDNA of these genes have now become publicly available. The company's mission statement is 'to become a leader in high throughput antibody and protein production'. In addition to generation of antibodies, the company has applications and systems which assist in gene synthesis, cytogenics, protein expression, and circulating tumor DNA. On the backs of their research on circulating tumor DNA, in 2018, Abnova filed an application titled 'Prenatal Detection of Fetal Trophoblasts in Maternal Circulation' which will allow the company to use its CytoQuest™ CR positive selection microfluidic system in order to 'capture and detect EVT in maternal peripheral blood'. The application states that the company's technology is successful in capturing 'EVT from material circulation via biotinylated HLA-G monoclonal antibody on a streptavidin coated CytoChipNano substrate'. Abnova has 'developed a proprietary trophoblast antigen (TBA) monoclonal antibody to detect and validate EVT after HLA-G monoclonal antibody capturing' which in noted on the application as the 'first known demonstration of EVT capture and detection using an antibody-based, positive selection microfluidic system'.
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