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Lab-on-a-chip technology: impacting non-invasive prenatal diagnostics (NIPD) through miniaturisation Chaitanya Kantak,*a Chia-Pin Chang,a Chee Chung Wong,a Aniza Mahyuddin,b Mahesh Choolanib and Abdur Rahmana This paper aims to provide a concise review of non-invasive prenatal diagnostics (NIPD) to the lab-on-a-chip and microfluidics community. Having a market of over one billion dollars to explore and a plethora of
Received 26th August 2013, Accepted 10th December 2013 DOI: 10.1039/c3lc50980j
applications, NIPD requires greater attention from microfluidics researchers. In this review, a complete overview of conventional diagnostic procedures including invasive as well as non-invasive (fetal cells and cell-free fetal DNA) types are discussed. Special focus is given to reviewing the recent and past microfluidic approaches to NIPD, as well as various commercial entities in NIPD. This review concludes with future
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challenges and ethical considerations of the field.
1. Introduction The first major discovery that laid the foundation of fetal diagnostics was in 1966 when Steele and Berg verified the presence of fetal cells in amniotic fluid. These were cultured to study the genetic nature of the fetus.1 This work was followed by successful development of invasive techniques in the clinical setting for gathering genetic information about the fetus during the early gestational period. Medical procedures such as amniocentesis and chorionic villi sampling were perfected to determine the presence of genetic abnormalities in fetuses. These disorders can be of different types: (1) common chromosome abnormalities such as Down syndrome (trisomy 21), Edward syndrome (trisomy 18), and Patau syndrome (trisomy 13) which are regarded as a type of aneuploidy; (2) aneuploidies related to X and Y chromosomes such as Triple X syndrome, Klinefelter syndrome and Turner syndrome; (3) single gene Mendelian disorders propagated through either or both parents, such as cystic fibrosis, hemoglobinopathies and neurodegenerative disorders; and (4) medical conditions such as a fetal rhesus D (RhD) positive genotype in a rhesus D negative mother, as well as cases of erythroblastosis fetalis. The majority of aneuploidic cases could lead to termination of the fetus and have miscarriage rates of up to 35%.2,3 Survival rates of 1 in 800, 1 in 6000, and 1 in 10 000 births have been reported with trisomies 21, 18 and 13 respectively.2 In such cases, parents have the choice of being prepared a
Institute of Microelectronics, Agency for Science Technology and Research, 11 Science Park Road, Singapore Science Park 2, Singapore 117685, Singapore. E-mail: [email protected]; Fax: +65 6773 1914; Tel: +65 6770 5638 b YLL School of Medicine, National University of Singapore, Singapore
This journal is © The Royal Society of Chemistry 2014
psychologically, financially and socially for the birth of a child with a medical pre-condition, as well as the necessity of family and social support, or to terminate the pregnancy.4 If the parents decide to terminate the pregnancy, it will be helpful to alleviate any physiological trauma and medical complications that may occur. While current non-invasive screening techniques have higher false positive rates, invasive procedures were reported to have complications leading to miscarriage, infections and even maternal fatality. A detailed explanation will be presented in section 2 of this review. To circumvent the aforementioned issues, clinical researchers were able to propose non-invasive prenatal diagnostic (NIPD) techniques in the last decade. These probe for the presence of cell-free fetal DNA (cffDNA) or fetalderived cells in the maternal blood (see detailed discussion in section 3). To date, microfluidic approaches have offered certain solutions for non-invasive prenatal diagnosis (section 5). However, most of these methods neither address the criteria of efficiency in blood isolation nor direct applicability in clinical settings. This review discusses the advantages as well as shortcomings of such approaches in detail. Lab on a Chip readers should also refer to an extensive review published in 2010 by Kavanagh et al.5 for a deeper understanding of conventional techniques of fetal cell isolation, as well as a discussion of microelectromechanical systems (MEMS)/microfluidics-based approaches. Section 6 of this manuscript briefly lists the commercial entities working in the nascent field of NIPD. The topic of NIPD is controversial because of political and religious opinions; therefore we have provided ethical considerations of NIPD to Lab on a Chip readers in this review (section 7.2). In this review, we intend to highlight developments in the field of NIPD in a broader sense by covering recent developments in
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Critical review
microfluidic isolation of fetal cells and microfluidics-based cell-free DNA NIPD techniques. We aim to bring the attention of the lab-on-a-chip community to this extremely challenging problem which can possibly be addressed using robust, miniaturised solutions which in turn will have a tremendous commercial impact on the market of prenatal diagnostics in the coming years. With the advancement of microfabrication methods and enhanced understanding of microfluidic principles, we believe that microfluidics will play a crucial role in defining the future of NIPD.
2. Gold standards of prenatal diagnostic (PD) techniques 2.1 Invasive PD techniques Prenatal genetic diagnostic procedures were introduced in the late 1960s.6 Since then, invasive techniques such as amniocentesis and chorionic villi sampling (CVS) have been established in the clinical setting. Amniocentesis is usually offered to women in advanced maternal age, which is 35 years and above. The procedure is usually carried out between the 15th and 22nd gestational weeks, wherein a needle is inserted through the abdominal and uterine walls into the amniotic sac to extract amniotic fluid. The amniocytes are isolated from the amniotic fluid and are observed for chromosomal abnormalities using molecular techniques such as Fluorescence In Situ Hybridization (FISH) or Polymerase Chain Reaction (PCR). The risk of miscarriage is reported to be up to 1 in 400 or less whenever the technique is carried out by experienced personnel.7 However, post-amniocentesis complications such as an intra-abdominal viscous injury or haemorrhage have been reported. In extremely rare cases, serious complications such as fulminant sepsis due to Escherichia coli or clostridia result in maternal mortality.8 Due to a higher rate of pregnancy loss, patients have been advised not to undergo amniocentesis before 13 weeks of gestation.9 Chorionic villus sampling (CVS) is another commonlypracticed invasive technique. This involves the sampling of chorionic villi from placental tissue during 10–12 weeks of gestation. CVS performed after 15 weeks of gestation has higher chances of causing miscarriage than amniocentesis.10 Although CVS provides gynecologists with similar information (such as chromosomal status, enzyme levels and genetic mutations) as that from amniocentesis, CVS samples cannot be used in assays involving alpha-fetoprotein.6 Additionally, the CVS procedure is more difficult to perform and requires highly skillful and experienced medical personnel when compared with amniocentesis.6 CVS procedures were reported to have caused fetal losses of approximately 5% in a study conducted by NICHD in the USA.11 Pregnancy terminations or abortions can have procedural complications leading to maternal fatalities. The rate of such fatalities is higher in the later stages of pregnancy. Abortions were reported to have caused maternal fatalities of 1 in 100 000 in the first trimester but 7–10 in 100 000 in the second
842 | Lab Chip, 2014, 14, 841–854
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trimester.12 This also highlights the need for early fetal diagnosis in order to reduce maternal fatalities. 2.2 Non-invasive maternal serum analyte and ultrasound screening Non-invasive screening tests are commonly carried out in the first or second, or even both trimesters. The first trimester screening tests (Triple Screen) are conducted between the 11th and 14th gestational weeks to detect the levels of biochemical markers, such as Pregnancy Associated Plasma Protein A (PAPP-A) and free beta Human Chorionic Gonadotropin (hCG), in the maternal serum. This blood test is accompanied by ultrasound screening for measuring nuchal translucency. A pregnancy associated with trisomy 21 is indicated by reduced levels of PAPP-A, increased levels of hCG, and an increase in the nuchal translucency. These tests generally suffer from false negatives and false positives and have a lower detection rate of 80–95%.6 In some cases, they require further investigation using invasive procedures. False positive rates of up to 5% were reported,13 limiting widespread adoption of these techniques as the screening tests of choice. The second-trimester screening (Quad Screen) is carried out by determining the levels of four biomarkers: alpha-fetoprotein (AFP), hCG, unconjugated oestriol (uE3), and dimeric inhibin-A. The detection rate for trisomy 21 using second trimester tests was found to be approximately 80% for women under and above the age of 35 years, with a false positive rate of 5%.6 The combined approach of carrying out screening in both trimesters as well as tests like CVS or amniocentesis, is employed in order to mitigate the risk of aneuploidic pregnancy.14 The current gold standards of prenatal diagnostics mainly suffer from: their invasive nature and complications leading to fetal or, rarely, maternal fatalities; requirement for highly skilled personnel to carry out amniocentesis and CVS; and high false positive and false negative rates leading to unexpected outcomes of pregnancies. The present technique of CVS is only capable of determining the genetic footprint of fetuses from the 10th to 12th weeks of gestation. On the other hand, NIPD procedures can be implemented as early as the 5th week of gestation after the onset of fetal cells/DNA in the maternal blood. One such example is of trophoblastic fetal cells, which are used to detect spinal muscular atrophy at the 5th week of pregnancy.15,16 Thus, NIPD techniques implemented in the early phase of the first trimester (Fig. 1) can possibly help to a great extent for clinical and parental decision-making. Such non-invasive techniques are discussed in the following section in detail.
3. NIPD – nucleated fetal cells and cell-free fetal DNA (cffDNA) in the maternal blood 3.1 Nucleated fetal cells Different types of nucleated fetal cells have been found to be circulating in the peripheral maternal blood. However, not all
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Fig. 1 A comparative schematic of prenatal diagnostic techniques and their applicability with respect to the progress of pregnancy: conventional invasive procedures (amniocentesis, chorionic villi sampling); serum screening techniques (triple and quad screens); and non-invasive prenatal diagnostics (circulating fetal cells and cell-free fetal DNA sampling methods).
of them are useful for non-invasive diagnosis of an on-going pregnancy. Four types of fetal nucleated cells have been reported: trophoblasts, fetal nucleated red blood cells, hematopoietic progenitor cells and lymphocytes. Trophoblasts or circulating fetal trophoblastic cells (CfTC) are considered to be an important cell type for NIPD. Trophoblasts are also the first cells which are known to cross into the maternal peripheral blood. These cells are difficult to detect in the maternal circulation as they are rapidly cleared by the pulmonary circulation and there are no commercially available antibodies targeting them with high specificity.17 Trophoblasts are known to have a size of 20 μm, have multiple nuclei and a cell boundary with rough morphology. Trophoblasts have been used in the detection of cystic fibrosis and spinal muscular atrophy in pregnancies.16,18,19 Fetal Nucleated Red Blood Cells (fNRBCs) are ideal for noninvasive prenatal diagnosis owing to their limited lifespan within maternal blood.20 They have a distinct morphology: a nucleus bearing a full complement of nuclear genes, having a diameter of 4–6 μm; a total cell size of 10–18 μm; and a cell membrane which is highly deformable. The fNRBCs express an intracellular globin marker called epsilon (ε+), a developmentspecific marker. Enrichment of fNRBCs from maternal blood is achieved using different antibodies such as anti-CD71, antiglycophorin A (GPA) or anti-CD36.21 However, the enrichment of fNRBCs is challenging because: (1) fNRBCs are extremely rare with a concentration of approximately one cell per milliliter of maternal blood; and (2) the enriched cells may contain up to 50% nucleated RBCs (NRBCs) of maternal origin.22 The technique of isolating fetal hematopoietic stem cells (fHSC) has not matured yet owing to their extremely low circulating numbers as well as lack of specific and consistent surface markers. The inherent proliferative nature of the stem cells could lead to misdiagnosis of the current pregnancy. Similar to fHSCs, fetal leukocytes have been detected in the maternal circulation even 27 years post-pregnancy.23 The enrichment techniques of fetal leukocytes are limited to the use of antibodies against paternally-derived human
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leukocyte antigens (HLA) from maternal blood, thus making prior HLA testing of both parents mandatory.24 Until now, fNRBCs remain one of the most extensively studied circulating fetal cells in comparison to trophoblasts, fHSCs and leukocytes. This is because of: (1) their peculiar size and shape which are distinctly different from those of white blood cells (WBCs); (2) the presence of molecular markers; and (3) the absence of a proliferative nature. These properties make fNRBCs the prime candidate for the development of isolation-type microfluidic devices. These methods will be discussed in later sections of this review.
3.2 Cell-free fetal DNA The isolation and analysis of cell-free fetal DNA from the maternal plasma has shown tremendous potential for noninvasive prenatal diagnosis. In contrast to nucleated fetal cells, cell-free DNA is present abundantly in the maternal plasma constituting up to approximately 10% of maternal DNA.25,26 However, genetic information is muddled in the fragments of fetal DNA, which are typically only hundreds of base pairs in length, rendering them difficult to decipher and requiring sophisticated molecular diagnostic techniques to retrieve fetal genetic information.27 Pioneering work by Lo et al.28,29 demonstrated that allelic ratios of placental specific mRNA could be used to determine trisomy 21. Honda et al. successfully showed detection of the Y-chromosome using real time PCR and demonstrated sensitivity of 100% as early as during the fifth gestational week.15 Cell-free fetal DNA can further facilitate the diagnosis of various chromosomal disorders such as aneuploidies, sex-linked disorders, beta-thalassemia, fetal rhesus D status and congenital adrenal hyperplasia.30–34 Most of the commercial enterprises in the field of NIPD are primarily based on cell-free fetal DNA analysis and are discussed in detail in section 6 of this review. The distinctive advantages and limitations of NIPD techniques, based on the isolation of cell-free fetal DNA and nucleated fetal cells are summarised in Table 1.
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Table 1 Comparative analysis of cell-free fetal DNA and fetal cells in NIPD
Cell-free fetal DNA Higher abundance in maternal plasma (>10%) Assay for trisomies 21, 18 and 13 have been developed Automated platform available Disadvantages Fragmented nature of fetal DNA High cost assay with lower throughput Specific assay needed to detect aneuploidy and genetic disorders
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Advantages
Fetal cells 25
3.3 Etiology of disorders in NIPD The presence of fetal abnormalities manifests itself in changes in the number of fetal cells or amount of cell-free fetal DNA in the peripheral maternal blood when compared to normal pregnancies. The usual count of fNRBCs detected in the first trimester of normal pregnancy is reported to be up to two cells per milliliter of whole blood.35,36 The total number of fNRBCs detected during aneuploidic pregnancies was reported to be higher than the number of fNRBCs detected during normal pregnancies.37,38 Elevated fetal DNA levels in the plasma are the basis for the diagnostic technique of cellfree fetal DNA analysis in cases of placental pathologies.39 Such etiologic changes have been observed quantitatively in the following abnormalities for nucleated fetal cells and cell-free fetal DNA respectively. Polyhydramnios is the condition where the amniotic fluid index is more than twice the standard deviation above the mean when measured in the late second to third trimesters. The cause of polyhydramnios could be due to various maternal and fetal conditions like diabetes mellitus, congenital anomalies, iso-immunization, multiple gestation and placental abnormalities.40 Zhong et al. reported an excessive increase in the population of NRBCs in polyhydramnios pregnancies at 34 weeks of gestation. In such cases, NRBC counts (~230), of both the mother and the fetus, were found to be higher than the counts in normal pregnancies (~5). This phenomenon was explained by the authors as the escaping of large numbers of fNRBCs through villi to maternal uterine veins owing to the increase in pressure on the placenta during polyhydramnios. Other etiologic examples are pre-eclampsia (PE) and intrauterine growth restriction (IUGR). These have been responsible for fetal and maternal mortality. The diagnostic biochemical markers correlating with the inflammatory response and endothelial dysfunction for PE can only be detected at the end of the second trimester, thus making it difficult to introduce intervention to prevent the disease. On the other hand, the correlation of PE and IUGR with the elevated levels of cell-free fetal DNA as early as 11–14 weeks of pregnancy was evident in the study by Illanes et al.41 The clearance of fetal DNA through renal pathways provides an opportunity to perform NIPD using maternal urine samples. However, past reports are inconclusive in determining its feasibility in early diagnosis. Some recent reports42 have noted the difficulty in detecting cffDNA in maternal urine due to contamination arising from traces of sperm DNA (e.g. occurrence of male DNA in cases of pregnancies
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Pure, complete fetal DNA can be obtained Whole genomic DNA amplification is possible Confirmation of fetal identity is possible in the 1st trimester Extreme rarity of cells (1 cell ml−1 of maternal blood)35 Automation is challenging Placental mosaicism within trophoblastic fetal cells17
with female foetuses) and quantities of cffDNA being lower than the detection limits.
4. Conventional approaches to NIPD Conventional means of isolating fNRBCs can be classified into five categories: (1) cell size-based; (2) density-based; (3) optical-based; (4) magnetic-based; and (5) adhesion-based. These separation methods can be used individually or in combination. Density- and size-based methods for separating cells are well-established. The most commonly used techniques are density gradient centrifugation (DGC)43 and sizebased filtration. Fetal cell researchers have used centrifugation on samples to reduce the amount of mature red blood cells (RBCs) and to enrich the mononuclear cells in order to identify their morphological characteristics. The mononuclear cells are characterized by immunocytochemical staining,44 May–Giemsa staining45 or the Pappenheim method.35 However, centrifugation methods pose a potential risk of infection to handlers due to aerosol generation, cause cell damage and result in an estimated cell loss of 30 to 50%.43 The technique is laborious, requires accurate balancing of sample tubes and is difficult to integrate into continuous flow systems. A filtration technique called “Isolation by Size of Epithelial Tumor Cells” (ISET) to isolate fetal trophoblasts was proposed by Vona et al.46 It was able to successfully separate trophoblasts for FISH as well as for DNA extraction and subsequent molecular analyses such as PCR. However, this method is not applicable as a standard NIPD technique due to the placental mosaic nature of trophoblast cells. Optical-based separation techniques include fluorescenceactivated cell sorting (FACS) and laser micro-dissection. Researchers have shown that FACS can be used to identify fetal cells by relying on the detection of specific cell surface antigens, such as CD71 (transferrin receptor), CD36 (thrombospondin receptor), and GPA (glycophorin A).47 Surface markers such as CD45, CD4, CD32, and CD19 can be used to remove unwanted cells such as WBCs.48,49 However, FACS set-ups usually consist of a laser source, optical components for light collection and detection, as well as fluidic and electrical components which altogether make the technique capital-intensive and bulky. FACS-based sorting techniques are tedious and require welltrained personnel for operation. Magnetic-based separation techniques can be applied in either of two ways: (a) coating paramagnetic or ferromagnetic particles with cell-targeting molecules to achieve cell
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separation; or (b) using the intrinsic magnetic moment of target molecules such as deoxygenated RBCs to achieve this. Magnetic-activated cell sorting (MACS) uses specific superparamagnetic bead-coated antibodies to coat target cells. A strong magnet is then applied to immobilize bead-coated cells while unlabeled cells are washed away. The immobilized cells can then be collected by removing the magnet. MACS is reported to have poorer yield and purity compared to FACS and can lead to sample contamination and cell damage due to shear forces. However, applying Ficoll gradient purification to the samples before conducting MACS can improve the selectivity for NRBCs.50 Adhesion-based separation was performed by Kitagawa et al.51 using glass slides coated with a galactose-containing polymer and soybean agglutinin (SBA), a galactose-specific lectin, bound to the polymer surface. As galactose molecules are highly expressed on the surface of erythroid precursor cells, NRBCs are enriched by their absorption to the slides when they pass by the surface and are captured by the SBA. However, screening the slides is labor-intensive and sample purity is not high due to high amounts of contaminating non-nucleated RBCs. Conventional processes to isolate fetal cells are laborious, time-consuming and difficult to automate. These problems could be overcome using microfluidic technology.
5. Microfluidics in NIPD 5.1 Microfluidic approaches using cell-free fetal DNA PCR reactions have been explored in microfluidic systems in detail. These have been developed in different materials like silicon, glass and plastic materials like polymethylacrylate (PMMA), polycarbonate (PC) and polydimethylsiloxane (PDMS).52–54 Extremely low sample and reagent volumes, higher surface area to volume ratios, and most importantly, higher sensitivity than conventional systems are the main advantages of microfluidics-based PCR devices.55 With the recent emergence of droplet microfluidic devices, the capability of performing thousands of compartmentalized, parallel PCR reactions in individual droplets has been imparted to microsystems.56 In digital PCR, DNA is quantified by counting the instances of amplification of each copy of DNA. The PCR mixture along with the DNA template is diluted and distributed within numerous compartments so that every compartment has less than one copy of DNA template and PCR products are detected by a fluorescence signal. Quantification is achieved by correlating the number of DNA templates to the number of compartments in which the signal is detected. This can be further used to detect copy number variations or single nucleotide polymorphisms (SNPs) in the DNA sequence. The automation of digital PCR within microfluidic devices has been explored in the past by Quake's group and has been made commercially available by Fluidigm®.57 Chipbased digital PCR is also offered by Life Technologies in the form of the OpenArray™ platform.58 This consists of microfabricated through-holes which utilize hydrophobic coatings
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to immobilize numerous droplets. Each array is capable of carrying out up to 36 000 parallel reactions. Another interesting microfluidic digital PCR approach, named SlipChip,59 was proposed by Ismagilov's group. This system consists of glass plates with alignable microfabricated features for compartmentalization of reactions isolated by oil-filled channels. The system was reported to have good sensitivity and dynamic range due to its multivolume approach. Quake and co-workers showed the non-invasive diagnosis of fetal aneuploidy of trisomies 21, 18 and 13 using microfluidic digital PCR, as early as after 14 gestational weeks.60 The length of fetal DNA fragments (~169 bp, Fig. 2A) was found to be shorter than that of the maternal DNA (>250 bp), which is in agreement with previous reports.61 The authors were able to successfully identify 9 cases of Down syndrome, 2 cases of Edward syndrome and a case of Patau Syndrome from a cohort of 18 patients. Interestingly, the findings of this work revealed that the majority of free DNA fragments obtained from the plasma shared features of nucleosomal DNA, further pointing out that the quantity of a particular locus may not be representative of the quantity of the entire chromosome. Fan et al. showed the successful identification of aneuploidies such as trisomy 21, trisomy 18, and trisomy 13 in ongoing pregnancies with comparable accuracy using the same microfluidic digital PCR platform.62 However, the samples were obtained by amniocentesis and CVS during pregnancy. This in turn shows that microfluidic digital PCR can be used either on its own as a technique to detect fetal aneuploidy non-invasively or integrated with current invasive diagnostic practices. Whale et al.63 recently found that the microfluidic digital PCR has better sensitivity than conventional quantitative PCR in detecting copy number variation (CNV), which is the change in the genomic DNA of an individual leading to an abnormal number of copies of cffDNA. Additionally, the authors pointed out that digital microfluidic PCR does not suffer from the issues of technical variability in quantitative PCR, commonly observed between different laboratories. Dennis Lo and co-workers64 also demonstrated that microfluidic digital PCR has the least quantitative bias for the measurement of DNA fragments, higher precision and higher clinical sensitivity compared with conventional non-digital real-time PCR. The authors also reported that ZFY/ZFX assays performed on the microfluidic digital PCR platform (Fig. 2B) showed that the median fractional concentration of fetal DNA in the maternal plasma was two times more than previously reported during all three trimesters of pregnancy. These recent reports show the significant improvements that can be brought to the field of NIPD using microfluidic digital PCR. Tsui et al. demonstrated the application of microfluidic digital PCR to the non-invasive detection of haemophilia by analysis of maternal plasma.65 A pregnant haemophilic mother has a 25% chance of carrying an affected male fetus. On the other hand 3–4% of fetuses suffer from cranial bleeding during labor and pregnancy. The authors suggested a noninvasive screening methodology for potential haemophilia
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Fig. 2 (A) Figure from Fan et al.60 showing the average fragment size of 170 bp for cffDNA. (B) From Lun et al. commercially available, microfluidic digital PCR offers higher sensitivity than the conventional RT-PCR.64 (Reproduced with permission from ref. 60 and ref. 64).
carrier pregnancies, and were able to successfully identify 12 clinical samples to be of a heterozygous nature for the causative mutations. The quantity of fetal DNA was determined using the ZFY/X assay and found to be less than 10% of the total concentration of cell-free DNA. Using the microfluidic digital PCR platform, Barrett et al.66 detected sickle cell anaemia in 82% of male fetuses and 75% of female fetuses. They analyzed the dosage of the variant encoding hemoglobin S (mutant) relative to that of hemoglobin A (wildtype) using cffDNA from maternal plasma samples. In addition to the microfluidic PCR platforms, Hahn and co-researchers67 had developed a dedicated microsystem to pre-concentrate and separate fetal DNA from maternal plasma samples. The platform was realised in PMMA and consisted of a polyethylene terephthalate membrane for electrokinetic trapping of cffDNA. The authors used a field-amplified sample stacking technique to concentrate the samples and were able to concentrate 80 μl of the input sample into a final volume of 2 μl, with the desired fraction of nucleic acids of targeted length. The microsystem was eventually validated by separating the DNA fragments (
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Lab-on-a-chip technology: impacting non-invasive prenatal diagnostics (NIPD) through miniaturisation Chaitanya Kantak,*a Chia-Pin Chang,a Chee Chung Wong,a Aniza Mahyuddin,b Mahesh Choolanib and Abdur Rahmana This paper aims to provide a concise review of non-invasive prenatal diagnostics (NIPD) to the lab-on-a-chip and microfluidics community. Having a market of over one billion dollars to explore and a plethora of
Received 26th August 2013, Accepted 10th December 2013 DOI: 10.1039/c3lc50980j
applications, NIPD requires greater attention from microfluidics researchers. In this review, a complete overview of conventional diagnostic procedures including invasive as well as non-invasive (fetal cells and cell-free fetal DNA) types are discussed. Special focus is given to reviewing the recent and past microfluidic approaches to NIPD, as well as various commercial entities in NIPD. This review concludes with future
www.rsc.org/loc
challenges and ethical considerations of the field.
1. Introduction The first major discovery that laid the foundation of fetal diagnostics was in 1966 when Steele and Berg verified the presence of fetal cells in amniotic fluid. These were cultured to study the genetic nature of the fetus.1 This work was followed by successful development of invasive techniques in the clinical setting for gathering genetic information about the fetus during the early gestational period. Medical procedures such as amniocentesis and chorionic villi sampling were perfected to determine the presence of genetic abnormalities in fetuses. These disorders can be of different types: (1) common chromosome abnormalities such as Down syndrome (trisomy 21), Edward syndrome (trisomy 18), and Patau syndrome (trisomy 13) which are regarded as a type of aneuploidy; (2) aneuploidies related to X and Y chromosomes such as Triple X syndrome, Klinefelter syndrome and Turner syndrome; (3) single gene Mendelian disorders propagated through either or both parents, such as cystic fibrosis, hemoglobinopathies and neurodegenerative disorders; and (4) medical conditions such as a fetal rhesus D (RhD) positive genotype in a rhesus D negative mother, as well as cases of erythroblastosis fetalis. The majority of aneuploidic cases could lead to termination of the fetus and have miscarriage rates of up to 35%.2,3 Survival rates of 1 in 800, 1 in 6000, and 1 in 10 000 births have been reported with trisomies 21, 18 and 13 respectively.2 In such cases, parents have the choice of being prepared a
Institute of Microelectronics, Agency for Science Technology and Research, 11 Science Park Road, Singapore Science Park 2, Singapore 117685, Singapore. E-mail: [email protected]; Fax: +65 6773 1914; Tel: +65 6770 5638 b YLL School of Medicine, National University of Singapore, Singapore
This journal is © The Royal Society of Chemistry 2014
psychologically, financially and socially for the birth of a child with a medical pre-condition, as well as the necessity of family and social support, or to terminate the pregnancy.4 If the parents decide to terminate the pregnancy, it will be helpful to alleviate any physiological trauma and medical complications that may occur. While current non-invasive screening techniques have higher false positive rates, invasive procedures were reported to have complications leading to miscarriage, infections and even maternal fatality. A detailed explanation will be presented in section 2 of this review. To circumvent the aforementioned issues, clinical researchers were able to propose non-invasive prenatal diagnostic (NIPD) techniques in the last decade. These probe for the presence of cell-free fetal DNA (cffDNA) or fetalderived cells in the maternal blood (see detailed discussion in section 3). To date, microfluidic approaches have offered certain solutions for non-invasive prenatal diagnosis (section 5). However, most of these methods neither address the criteria of efficiency in blood isolation nor direct applicability in clinical settings. This review discusses the advantages as well as shortcomings of such approaches in detail. Lab on a Chip readers should also refer to an extensive review published in 2010 by Kavanagh et al.5 for a deeper understanding of conventional techniques of fetal cell isolation, as well as a discussion of microelectromechanical systems (MEMS)/microfluidics-based approaches. Section 6 of this manuscript briefly lists the commercial entities working in the nascent field of NIPD. The topic of NIPD is controversial because of political and religious opinions; therefore we have provided ethical considerations of NIPD to Lab on a Chip readers in this review (section 7.2). In this review, we intend to highlight developments in the field of NIPD in a broader sense by covering recent developments in
Lab Chip, 2014, 14, 841–854 | 841
View Article Online
Published on 23 January 2014. Downloaded by Yale University Library on 30/09/2014 11:59:56.
Critical review
microfluidic isolation of fetal cells and microfluidics-based cell-free DNA NIPD techniques. We aim to bring the attention of the lab-on-a-chip community to this extremely challenging problem which can possibly be addressed using robust, miniaturised solutions which in turn will have a tremendous commercial impact on the market of prenatal diagnostics in the coming years. With the advancement of microfabrication methods and enhanced understanding of microfluidic principles, we believe that microfluidics will play a crucial role in defining the future of NIPD.
2. Gold standards of prenatal diagnostic (PD) techniques 2.1 Invasive PD techniques Prenatal genetic diagnostic procedures were introduced in the late 1960s.6 Since then, invasive techniques such as amniocentesis and chorionic villi sampling (CVS) have been established in the clinical setting. Amniocentesis is usually offered to women in advanced maternal age, which is 35 years and above. The procedure is usually carried out between the 15th and 22nd gestational weeks, wherein a needle is inserted through the abdominal and uterine walls into the amniotic sac to extract amniotic fluid. The amniocytes are isolated from the amniotic fluid and are observed for chromosomal abnormalities using molecular techniques such as Fluorescence In Situ Hybridization (FISH) or Polymerase Chain Reaction (PCR). The risk of miscarriage is reported to be up to 1 in 400 or less whenever the technique is carried out by experienced personnel.7 However, post-amniocentesis complications such as an intra-abdominal viscous injury or haemorrhage have been reported. In extremely rare cases, serious complications such as fulminant sepsis due to Escherichia coli or clostridia result in maternal mortality.8 Due to a higher rate of pregnancy loss, patients have been advised not to undergo amniocentesis before 13 weeks of gestation.9 Chorionic villus sampling (CVS) is another commonlypracticed invasive technique. This involves the sampling of chorionic villi from placental tissue during 10–12 weeks of gestation. CVS performed after 15 weeks of gestation has higher chances of causing miscarriage than amniocentesis.10 Although CVS provides gynecologists with similar information (such as chromosomal status, enzyme levels and genetic mutations) as that from amniocentesis, CVS samples cannot be used in assays involving alpha-fetoprotein.6 Additionally, the CVS procedure is more difficult to perform and requires highly skillful and experienced medical personnel when compared with amniocentesis.6 CVS procedures were reported to have caused fetal losses of approximately 5% in a study conducted by NICHD in the USA.11 Pregnancy terminations or abortions can have procedural complications leading to maternal fatalities. The rate of such fatalities is higher in the later stages of pregnancy. Abortions were reported to have caused maternal fatalities of 1 in 100 000 in the first trimester but 7–10 in 100 000 in the second
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trimester.12 This also highlights the need for early fetal diagnosis in order to reduce maternal fatalities. 2.2 Non-invasive maternal serum analyte and ultrasound screening Non-invasive screening tests are commonly carried out in the first or second, or even both trimesters. The first trimester screening tests (Triple Screen) are conducted between the 11th and 14th gestational weeks to detect the levels of biochemical markers, such as Pregnancy Associated Plasma Protein A (PAPP-A) and free beta Human Chorionic Gonadotropin (hCG), in the maternal serum. This blood test is accompanied by ultrasound screening for measuring nuchal translucency. A pregnancy associated with trisomy 21 is indicated by reduced levels of PAPP-A, increased levels of hCG, and an increase in the nuchal translucency. These tests generally suffer from false negatives and false positives and have a lower detection rate of 80–95%.6 In some cases, they require further investigation using invasive procedures. False positive rates of up to 5% were reported,13 limiting widespread adoption of these techniques as the screening tests of choice. The second-trimester screening (Quad Screen) is carried out by determining the levels of four biomarkers: alpha-fetoprotein (AFP), hCG, unconjugated oestriol (uE3), and dimeric inhibin-A. The detection rate for trisomy 21 using second trimester tests was found to be approximately 80% for women under and above the age of 35 years, with a false positive rate of 5%.6 The combined approach of carrying out screening in both trimesters as well as tests like CVS or amniocentesis, is employed in order to mitigate the risk of aneuploidic pregnancy.14 The current gold standards of prenatal diagnostics mainly suffer from: their invasive nature and complications leading to fetal or, rarely, maternal fatalities; requirement for highly skilled personnel to carry out amniocentesis and CVS; and high false positive and false negative rates leading to unexpected outcomes of pregnancies. The present technique of CVS is only capable of determining the genetic footprint of fetuses from the 10th to 12th weeks of gestation. On the other hand, NIPD procedures can be implemented as early as the 5th week of gestation after the onset of fetal cells/DNA in the maternal blood. One such example is of trophoblastic fetal cells, which are used to detect spinal muscular atrophy at the 5th week of pregnancy.15,16 Thus, NIPD techniques implemented in the early phase of the first trimester (Fig. 1) can possibly help to a great extent for clinical and parental decision-making. Such non-invasive techniques are discussed in the following section in detail.
3. NIPD – nucleated fetal cells and cell-free fetal DNA (cffDNA) in the maternal blood 3.1 Nucleated fetal cells Different types of nucleated fetal cells have been found to be circulating in the peripheral maternal blood. However, not all
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Fig. 1 A comparative schematic of prenatal diagnostic techniques and their applicability with respect to the progress of pregnancy: conventional invasive procedures (amniocentesis, chorionic villi sampling); serum screening techniques (triple and quad screens); and non-invasive prenatal diagnostics (circulating fetal cells and cell-free fetal DNA sampling methods).
of them are useful for non-invasive diagnosis of an on-going pregnancy. Four types of fetal nucleated cells have been reported: trophoblasts, fetal nucleated red blood cells, hematopoietic progenitor cells and lymphocytes. Trophoblasts or circulating fetal trophoblastic cells (CfTC) are considered to be an important cell type for NIPD. Trophoblasts are also the first cells which are known to cross into the maternal peripheral blood. These cells are difficult to detect in the maternal circulation as they are rapidly cleared by the pulmonary circulation and there are no commercially available antibodies targeting them with high specificity.17 Trophoblasts are known to have a size of 20 μm, have multiple nuclei and a cell boundary with rough morphology. Trophoblasts have been used in the detection of cystic fibrosis and spinal muscular atrophy in pregnancies.16,18,19 Fetal Nucleated Red Blood Cells (fNRBCs) are ideal for noninvasive prenatal diagnosis owing to their limited lifespan within maternal blood.20 They have a distinct morphology: a nucleus bearing a full complement of nuclear genes, having a diameter of 4–6 μm; a total cell size of 10–18 μm; and a cell membrane which is highly deformable. The fNRBCs express an intracellular globin marker called epsilon (ε+), a developmentspecific marker. Enrichment of fNRBCs from maternal blood is achieved using different antibodies such as anti-CD71, antiglycophorin A (GPA) or anti-CD36.21 However, the enrichment of fNRBCs is challenging because: (1) fNRBCs are extremely rare with a concentration of approximately one cell per milliliter of maternal blood; and (2) the enriched cells may contain up to 50% nucleated RBCs (NRBCs) of maternal origin.22 The technique of isolating fetal hematopoietic stem cells (fHSC) has not matured yet owing to their extremely low circulating numbers as well as lack of specific and consistent surface markers. The inherent proliferative nature of the stem cells could lead to misdiagnosis of the current pregnancy. Similar to fHSCs, fetal leukocytes have been detected in the maternal circulation even 27 years post-pregnancy.23 The enrichment techniques of fetal leukocytes are limited to the use of antibodies against paternally-derived human
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leukocyte antigens (HLA) from maternal blood, thus making prior HLA testing of both parents mandatory.24 Until now, fNRBCs remain one of the most extensively studied circulating fetal cells in comparison to trophoblasts, fHSCs and leukocytes. This is because of: (1) their peculiar size and shape which are distinctly different from those of white blood cells (WBCs); (2) the presence of molecular markers; and (3) the absence of a proliferative nature. These properties make fNRBCs the prime candidate for the development of isolation-type microfluidic devices. These methods will be discussed in later sections of this review.
3.2 Cell-free fetal DNA The isolation and analysis of cell-free fetal DNA from the maternal plasma has shown tremendous potential for noninvasive prenatal diagnosis. In contrast to nucleated fetal cells, cell-free DNA is present abundantly in the maternal plasma constituting up to approximately 10% of maternal DNA.25,26 However, genetic information is muddled in the fragments of fetal DNA, which are typically only hundreds of base pairs in length, rendering them difficult to decipher and requiring sophisticated molecular diagnostic techniques to retrieve fetal genetic information.27 Pioneering work by Lo et al.28,29 demonstrated that allelic ratios of placental specific mRNA could be used to determine trisomy 21. Honda et al. successfully showed detection of the Y-chromosome using real time PCR and demonstrated sensitivity of 100% as early as during the fifth gestational week.15 Cell-free fetal DNA can further facilitate the diagnosis of various chromosomal disorders such as aneuploidies, sex-linked disorders, beta-thalassemia, fetal rhesus D status and congenital adrenal hyperplasia.30–34 Most of the commercial enterprises in the field of NIPD are primarily based on cell-free fetal DNA analysis and are discussed in detail in section 6 of this review. The distinctive advantages and limitations of NIPD techniques, based on the isolation of cell-free fetal DNA and nucleated fetal cells are summarised in Table 1.
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Table 1 Comparative analysis of cell-free fetal DNA and fetal cells in NIPD
Cell-free fetal DNA Higher abundance in maternal plasma (>10%) Assay for trisomies 21, 18 and 13 have been developed Automated platform available Disadvantages Fragmented nature of fetal DNA High cost assay with lower throughput Specific assay needed to detect aneuploidy and genetic disorders
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Fetal cells 25
3.3 Etiology of disorders in NIPD The presence of fetal abnormalities manifests itself in changes in the number of fetal cells or amount of cell-free fetal DNA in the peripheral maternal blood when compared to normal pregnancies. The usual count of fNRBCs detected in the first trimester of normal pregnancy is reported to be up to two cells per milliliter of whole blood.35,36 The total number of fNRBCs detected during aneuploidic pregnancies was reported to be higher than the number of fNRBCs detected during normal pregnancies.37,38 Elevated fetal DNA levels in the plasma are the basis for the diagnostic technique of cellfree fetal DNA analysis in cases of placental pathologies.39 Such etiologic changes have been observed quantitatively in the following abnormalities for nucleated fetal cells and cell-free fetal DNA respectively. Polyhydramnios is the condition where the amniotic fluid index is more than twice the standard deviation above the mean when measured in the late second to third trimesters. The cause of polyhydramnios could be due to various maternal and fetal conditions like diabetes mellitus, congenital anomalies, iso-immunization, multiple gestation and placental abnormalities.40 Zhong et al. reported an excessive increase in the population of NRBCs in polyhydramnios pregnancies at 34 weeks of gestation. In such cases, NRBC counts (~230), of both the mother and the fetus, were found to be higher than the counts in normal pregnancies (~5). This phenomenon was explained by the authors as the escaping of large numbers of fNRBCs through villi to maternal uterine veins owing to the increase in pressure on the placenta during polyhydramnios. Other etiologic examples are pre-eclampsia (PE) and intrauterine growth restriction (IUGR). These have been responsible for fetal and maternal mortality. The diagnostic biochemical markers correlating with the inflammatory response and endothelial dysfunction for PE can only be detected at the end of the second trimester, thus making it difficult to introduce intervention to prevent the disease. On the other hand, the correlation of PE and IUGR with the elevated levels of cell-free fetal DNA as early as 11–14 weeks of pregnancy was evident in the study by Illanes et al.41 The clearance of fetal DNA through renal pathways provides an opportunity to perform NIPD using maternal urine samples. However, past reports are inconclusive in determining its feasibility in early diagnosis. Some recent reports42 have noted the difficulty in detecting cffDNA in maternal urine due to contamination arising from traces of sperm DNA (e.g. occurrence of male DNA in cases of pregnancies
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Pure, complete fetal DNA can be obtained Whole genomic DNA amplification is possible Confirmation of fetal identity is possible in the 1st trimester Extreme rarity of cells (1 cell ml−1 of maternal blood)35 Automation is challenging Placental mosaicism within trophoblastic fetal cells17
with female foetuses) and quantities of cffDNA being lower than the detection limits.
4. Conventional approaches to NIPD Conventional means of isolating fNRBCs can be classified into five categories: (1) cell size-based; (2) density-based; (3) optical-based; (4) magnetic-based; and (5) adhesion-based. These separation methods can be used individually or in combination. Density- and size-based methods for separating cells are well-established. The most commonly used techniques are density gradient centrifugation (DGC)43 and sizebased filtration. Fetal cell researchers have used centrifugation on samples to reduce the amount of mature red blood cells (RBCs) and to enrich the mononuclear cells in order to identify their morphological characteristics. The mononuclear cells are characterized by immunocytochemical staining,44 May–Giemsa staining45 or the Pappenheim method.35 However, centrifugation methods pose a potential risk of infection to handlers due to aerosol generation, cause cell damage and result in an estimated cell loss of 30 to 50%.43 The technique is laborious, requires accurate balancing of sample tubes and is difficult to integrate into continuous flow systems. A filtration technique called “Isolation by Size of Epithelial Tumor Cells” (ISET) to isolate fetal trophoblasts was proposed by Vona et al.46 It was able to successfully separate trophoblasts for FISH as well as for DNA extraction and subsequent molecular analyses such as PCR. However, this method is not applicable as a standard NIPD technique due to the placental mosaic nature of trophoblast cells. Optical-based separation techniques include fluorescenceactivated cell sorting (FACS) and laser micro-dissection. Researchers have shown that FACS can be used to identify fetal cells by relying on the detection of specific cell surface antigens, such as CD71 (transferrin receptor), CD36 (thrombospondin receptor), and GPA (glycophorin A).47 Surface markers such as CD45, CD4, CD32, and CD19 can be used to remove unwanted cells such as WBCs.48,49 However, FACS set-ups usually consist of a laser source, optical components for light collection and detection, as well as fluidic and electrical components which altogether make the technique capital-intensive and bulky. FACS-based sorting techniques are tedious and require welltrained personnel for operation. Magnetic-based separation techniques can be applied in either of two ways: (a) coating paramagnetic or ferromagnetic particles with cell-targeting molecules to achieve cell
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separation; or (b) using the intrinsic magnetic moment of target molecules such as deoxygenated RBCs to achieve this. Magnetic-activated cell sorting (MACS) uses specific superparamagnetic bead-coated antibodies to coat target cells. A strong magnet is then applied to immobilize bead-coated cells while unlabeled cells are washed away. The immobilized cells can then be collected by removing the magnet. MACS is reported to have poorer yield and purity compared to FACS and can lead to sample contamination and cell damage due to shear forces. However, applying Ficoll gradient purification to the samples before conducting MACS can improve the selectivity for NRBCs.50 Adhesion-based separation was performed by Kitagawa et al.51 using glass slides coated with a galactose-containing polymer and soybean agglutinin (SBA), a galactose-specific lectin, bound to the polymer surface. As galactose molecules are highly expressed on the surface of erythroid precursor cells, NRBCs are enriched by their absorption to the slides when they pass by the surface and are captured by the SBA. However, screening the slides is labor-intensive and sample purity is not high due to high amounts of contaminating non-nucleated RBCs. Conventional processes to isolate fetal cells are laborious, time-consuming and difficult to automate. These problems could be overcome using microfluidic technology.
5. Microfluidics in NIPD 5.1 Microfluidic approaches using cell-free fetal DNA PCR reactions have been explored in microfluidic systems in detail. These have been developed in different materials like silicon, glass and plastic materials like polymethylacrylate (PMMA), polycarbonate (PC) and polydimethylsiloxane (PDMS).52–54 Extremely low sample and reagent volumes, higher surface area to volume ratios, and most importantly, higher sensitivity than conventional systems are the main advantages of microfluidics-based PCR devices.55 With the recent emergence of droplet microfluidic devices, the capability of performing thousands of compartmentalized, parallel PCR reactions in individual droplets has been imparted to microsystems.56 In digital PCR, DNA is quantified by counting the instances of amplification of each copy of DNA. The PCR mixture along with the DNA template is diluted and distributed within numerous compartments so that every compartment has less than one copy of DNA template and PCR products are detected by a fluorescence signal. Quantification is achieved by correlating the number of DNA templates to the number of compartments in which the signal is detected. This can be further used to detect copy number variations or single nucleotide polymorphisms (SNPs) in the DNA sequence. The automation of digital PCR within microfluidic devices has been explored in the past by Quake's group and has been made commercially available by Fluidigm®.57 Chipbased digital PCR is also offered by Life Technologies in the form of the OpenArray™ platform.58 This consists of microfabricated through-holes which utilize hydrophobic coatings
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to immobilize numerous droplets. Each array is capable of carrying out up to 36 000 parallel reactions. Another interesting microfluidic digital PCR approach, named SlipChip,59 was proposed by Ismagilov's group. This system consists of glass plates with alignable microfabricated features for compartmentalization of reactions isolated by oil-filled channels. The system was reported to have good sensitivity and dynamic range due to its multivolume approach. Quake and co-workers showed the non-invasive diagnosis of fetal aneuploidy of trisomies 21, 18 and 13 using microfluidic digital PCR, as early as after 14 gestational weeks.60 The length of fetal DNA fragments (~169 bp, Fig. 2A) was found to be shorter than that of the maternal DNA (>250 bp), which is in agreement with previous reports.61 The authors were able to successfully identify 9 cases of Down syndrome, 2 cases of Edward syndrome and a case of Patau Syndrome from a cohort of 18 patients. Interestingly, the findings of this work revealed that the majority of free DNA fragments obtained from the plasma shared features of nucleosomal DNA, further pointing out that the quantity of a particular locus may not be representative of the quantity of the entire chromosome. Fan et al. showed the successful identification of aneuploidies such as trisomy 21, trisomy 18, and trisomy 13 in ongoing pregnancies with comparable accuracy using the same microfluidic digital PCR platform.62 However, the samples were obtained by amniocentesis and CVS during pregnancy. This in turn shows that microfluidic digital PCR can be used either on its own as a technique to detect fetal aneuploidy non-invasively or integrated with current invasive diagnostic practices. Whale et al.63 recently found that the microfluidic digital PCR has better sensitivity than conventional quantitative PCR in detecting copy number variation (CNV), which is the change in the genomic DNA of an individual leading to an abnormal number of copies of cffDNA. Additionally, the authors pointed out that digital microfluidic PCR does not suffer from the issues of technical variability in quantitative PCR, commonly observed between different laboratories. Dennis Lo and co-workers64 also demonstrated that microfluidic digital PCR has the least quantitative bias for the measurement of DNA fragments, higher precision and higher clinical sensitivity compared with conventional non-digital real-time PCR. The authors also reported that ZFY/ZFX assays performed on the microfluidic digital PCR platform (Fig. 2B) showed that the median fractional concentration of fetal DNA in the maternal plasma was two times more than previously reported during all three trimesters of pregnancy. These recent reports show the significant improvements that can be brought to the field of NIPD using microfluidic digital PCR. Tsui et al. demonstrated the application of microfluidic digital PCR to the non-invasive detection of haemophilia by analysis of maternal plasma.65 A pregnant haemophilic mother has a 25% chance of carrying an affected male fetus. On the other hand 3–4% of fetuses suffer from cranial bleeding during labor and pregnancy. The authors suggested a noninvasive screening methodology for potential haemophilia
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Fig. 2 (A) Figure from Fan et al.60 showing the average fragment size of 170 bp for cffDNA. (B) From Lun et al. commercially available, microfluidic digital PCR offers higher sensitivity than the conventional RT-PCR.64 (Reproduced with permission from ref. 60 and ref. 64).
carrier pregnancies, and were able to successfully identify 12 clinical samples to be of a heterozygous nature for the causative mutations. The quantity of fetal DNA was determined using the ZFY/X assay and found to be less than 10% of the total concentration of cell-free DNA. Using the microfluidic digital PCR platform, Barrett et al.66 detected sickle cell anaemia in 82% of male fetuses and 75% of female fetuses. They analyzed the dosage of the variant encoding hemoglobin S (mutant) relative to that of hemoglobin A (wildtype) using cffDNA from maternal plasma samples. In addition to the microfluidic PCR platforms, Hahn and co-researchers67 had developed a dedicated microsystem to pre-concentrate and separate fetal DNA from maternal plasma samples. The platform was realised in PMMA and consisted of a polyethylene terephthalate membrane for electrokinetic trapping of cffDNA. The authors used a field-amplified sample stacking technique to concentrate the samples and were able to concentrate 80 μl of the input sample into a final volume of 2 μl, with the desired fraction of nucleic acids of targeted length. The microsystem was eventually validated by separating the DNA fragments (
