Lab-on-a-chip technology: impacting non-invasive prenatal diagnostics (NIPD) through miniaturisation

Lab on a Chip

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CRITICAL REVIEW View Article OnlineView Journal | View Issue

Lab CThis journal is © The Royal Society of Chemistry 2014

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 5638b YLL School of Medicine, National University of Singapore, Singapore

Cite this: Lab Chip, 2014, 14, 841

Received 26th August 2013,Accepted 10th December 2013

DOI: 10.1039/c3lc50980j

www.rsc.org/loc

Lab-on-a-chip technology: impactingnon-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

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

challenges and ethical considerations of the field.

1. Introduction

The first major discovery that laid the foundation of fetaldiagnostics was in 1966 when Steele and Berg verified thepresence of fetal cells in amniotic fluid. These were culturedto study the genetic nature of the fetus.1 This work wasfollowed by successful development of invasive techniques inthe clinical setting for gathering genetic information aboutthe fetus during the early gestational period. Medical proce-dures such as amniocentesis and chorionic villi samplingwere perfected to determine the presence of genetic abnor-malities in fetuses. These disorders can be of different types:(1) common chromosome abnormalities such as Down syn-drome (trisomy 21), Edward syndrome (trisomy 18), and Patausyndrome (trisomy 13) which are regarded as a type of aneu-ploidy; (2) aneuploidies related to X and Y chromosomes suchas Triple X syndrome, Klinefelter syndrome and Turner syn-drome; (3) single gene Mendelian disorders propagated througheither or both parents, such as cystic fibrosis, hemoglobinopa-thies and neurodegenerative disorders; and (4) medical condi-tions such as a fetal rhesus D (RhD) positive genotype in arhesus D negative mother, as well as cases of erythroblastosisfetalis. The majority of aneuploidic cases could lead to termina-tion 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 birthshave been reported with trisomies 21, 18 and 13 respectively.2

In such cases, parents have the choice of being prepared

psychologically, financially and socially for the birth of a childwith a medical pre-condition, as well as the necessity of familyand social support, or to terminate the pregnancy.4 If the par-ents decide to terminate the pregnancy, it will be helpful toalleviate any physiological trauma and medical complicationsthat may occur.

While current non-invasive screening techniques havehigher false positive rates, invasive procedures were reported tohave complications leading to miscarriage, infections and evenmaternal fatality. A detailed explanation will be presented insection 2 of this review. To circumvent the aforementionedissues, clinical researchers were able to propose non-invasiveprenatal diagnostic (NIPD) techniques in the last decade. Theseprobe for the presence of cell-free fetal DNA (cffDNA) or fetal-derived cells in the maternal blood (see detailed discussion insection 3). To date, microfluidic approaches have offered cer-tain solutions for non-invasive prenatal diagnosis (section 5).However, most of these methods neither address the criteria ofefficiency in blood isolation nor direct applicability in clinicalsettings. This review discusses the advantages as well as short-comings of such approaches in detail. Lab on a Chip readersshould also refer to an extensive review published in 2010 byKavanagh et al.5 for a deeper understanding of conventionaltechniques of fetal cell isolation, as well as a discussion ofmicroelectromechanical systems (MEMS)/microfluidics-basedapproaches. Section 6 of this manuscript briefly lists the com-mercial entities working in the nascent field of NIPD. The topicof NIPD is controversial because of political and religiousopinions; therefore we have provided ethical considerations ofNIPD to Lab on a Chip readers in this review (section 7.2). Inthis review, we intend to highlight developments in the field ofNIPD in a broader sense by covering recent developments in

hip, 2014, 14, 841–854 | 841

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Lab on a ChipCritical review

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microfluidic isolation of fetal cells and microfluidics-basedcell-free DNA NIPD techniques. We aim to bring the attentionof the lab-on-a-chip community to this extremely challengingproblem which can possibly be addressed using robust,miniaturised solutions which in turn will have a tremendouscommercial impact on the market of prenatal diagnostics inthe coming years. With the advancement of microfabricationmethods and enhanced understanding of microfluidic princi-ples, we believe that microfluidics will play a crucial role indefining the future of NIPD.

2. Gold standards of prenataldiagnostic (PD) techniques2.1 Invasive PD techniques

Prenatal genetic diagnostic procedures were introduced in thelate 1960s.6 Since then, invasive techniques such as amnio-centesis and chorionic villi sampling (CVS) have beenestablished in the clinical setting. Amniocentesis is usuallyoffered to women in advanced maternal age, which is 35 yearsand above. The procedure is usually carried out between the15th and 22nd gestational weeks, wherein a needle is insertedthrough the abdominal and uterine walls into the amnioticsac to extract amniotic fluid. The amniocytes are isolatedfrom the amniotic fluid and are observed for chromosomalabnormalities using molecular techniques such as Fluores-cence In Situ Hybridization (FISH) or Polymerase Chain Reac-tion (PCR). The risk of miscarriage is reported to be up to 1 in400 or less whenever the technique is carried out by experi-enced personnel.7 However, post-amniocentesis complicationssuch as an intra-abdominal viscous injury or haemorrhage havebeen reported. In extremely rare cases, serious complicationssuch as fulminant sepsis due to Escherichia coli or clostridiaresult in maternal mortality.8 Due to a higher rate of preg-nancy loss, patients have been advised not to undergo amnio-centesis before 13 weeks of gestation.9

Chorionic villus sampling (CVS) is another commonly-practiced invasive technique. This involves the sampling ofchorionic villi from placental tissue during 10–12 weeks ofgestation. CVS performed after 15 weeks of gestation hashigher chances of causing miscarriage than amniocentesis.10

Although CVS provides gynecologists with similar informa-tion (such as chromosomal status, enzyme levels and geneticmutations) as that from amniocentesis, CVS samples cannotbe used in assays involving alpha-fetoprotein.6 Additionally,the CVS procedure is more difficult to perform and requireshighly skillful and experienced medical personnel when com-pared with amniocentesis.6 CVS procedures were reported tohave caused fetal losses of approximately 5% in a studyconducted by NICHD in the USA.11

Pregnancy terminations or abortions can have proceduralcomplications leading to maternal fatalities. The rate of suchfatalities is higher in the later stages of pregnancy. Abortionswere reported to have caused maternal fatalities of 1 in 100000in the first trimester but 7–10 in 100 000 in the second

842 | Lab Chip, 2014, 14, 841–854

trimester.12 This also highlights the need for early fetal diag-nosis in order to reduce maternal fatalities.

2.2 Non-invasive maternal serum analyteand ultrasound screening

Non-invasive screening tests are commonly carried out in thefirst or second, or even both trimesters. The first trimesterscreening tests (Triple Screen) are conducted between the11th and 14th gestational weeks to detect the levels of bio-chemical markers, such as Pregnancy Associated Plasma Pro-tein A (PAPP-A) and free beta Human Chorionic Gonadotropin(hCG), in the maternal serum. This blood test is accompaniedby ultrasound screening for measuring nuchal translucency. Apregnancy associated with trisomy 21 is indicated by reducedlevels of PAPP-A, increased levels of hCG, and an increase inthe nuchal translucency. These tests generally suffer fromfalse negatives and false positives and have a lower detectionrate of 80–95%.6 In some cases, they require further investiga-tion using invasive procedures. False positive rates of up to5% were reported,13 limiting widespread adoption of thesetechniques as the screening tests of choice.

The second-trimester screening (Quad Screen) is carried outby 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 testswas found to be approximately 80% for women under and abovethe age of 35 years, with a false positive rate of 5%.6 The com-bined approach of carrying out screening in both trimesters aswell as tests like CVS or amniocentesis, is employed in order tomitigate the risk of aneuploidic pregnancy.14

The current gold standards of prenatal diagnostics mainlysuffer from: their invasive nature and complications leadingto fetal or, rarely, maternal fatalities; requirement for highlyskilled personnel to carry out amniocentesis and CVS; andhigh false positive and false negative rates leading to unex-pected outcomes of pregnancies. The present technique ofCVS is only capable of determining the genetic footprint offetuses from the 10th to 12th weeks of gestation. On theother hand, NIPD procedures can be implemented as early asthe 5th week of gestation after the onset of fetal cells/DNA inthe maternal blood. One such example is of trophoblasticfetal cells, which are used to detect spinal muscular atrophyat the 5th week of pregnancy.15,16 Thus, NIPD techniquesimplemented in the early phase of the first trimester (Fig. 1)can possibly help to a great extent for clinical and parentaldecision-making. Such non-invasive techniques are discussedin the following section in detail.

3. NIPD – nucleated fetal cells andcell-free fetal DNA (cffDNA) in thematernal blood3.1 Nucleated fetal cells

Different types of nucleated fetal cells have been found to becirculating in the peripheral maternal blood. However, not all

This journal is © The Royal Society of Chemistry 2014


Fig. 1 A comparative schematic of prenatal diagnostic techniques and their applicability with respect to the progress of pregnancy: conventionalinvasive procedures (amniocentesis, chorionic villi sampling); serum screening techniques (triple and quad screens); and non-invasive prenataldiagnostics (circulating fetal cells and cell-free fetal DNA sampling methods).

Lab on a Chip Critical review

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of them are useful for non-invasive diagnosis of an on-goingpregnancy. Four types of fetal nucleated cells have beenreported: trophoblasts, fetal nucleated red blood cells, hema-topoietic progenitor cells and lymphocytes.

Trophoblasts or circulating fetal trophoblastic cells (CfTC)are considered to be an important cell type for NIPD. Tropho-blasts are also the first cells which are known to cross intothe maternal peripheral blood. These cells are difficult todetect in the maternal circulation as they are rapidly clearedby the pulmonary circulation and there are no commerciallyavailable antibodies targeting them with high specificity.17

Trophoblasts are known to have a size of 20 μm, have multi-ple nuclei and a cell boundary with rough morphology. Tro-phoblasts have been used in the detection of cystic fibrosisand spinal muscular atrophy in pregnancies.16,18,19

Fetal Nucleated Red Blood Cells (fNRBCs) are ideal for non-invasive prenatal diagnosis owing to their limited lifespanwithin maternal blood.20 They have a distinct morphology: anucleus bearing a full complement of nuclear genes, having adiameter of 4–6 μm; a total cell size of 10–18 μm; and a cellmembrane which is highly deformable. The fNRBCs express anintracellular globin marker called epsilon (ε+), a development-specific marker. Enrichment of fNRBCs from maternal blood isachieved using different antibodies such as anti-CD71, anti-glycophorin A (GPA) or anti-CD36.21 However, the enrichmentof fNRBCs is challenging because: (1) fNRBCs are extremelyrare with a concentration of approximately one cell per millili-ter of maternal blood; and (2) the enriched cells may containup 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 cir-culating numbers as well as lack of specific and consistentsurface markers. The inherent proliferative nature of thestem cells could lead to misdiagnosis of the current preg-nancy. Similar to fHSCs, fetal leukocytes have been detectedin the maternal circulation even 27 years post-pregnancy.23

The enrichment techniques of fetal leukocytes are limitedto the use of antibodies against paternally-derived human


leukocyte antigens (HLA) from maternal blood, thus makingprior HLA testing of both parents mandatory.24

Until now, fNRBCs remain one of the most extensivelystudied circulating fetal cells in comparison to trophoblasts,fHSCs and leukocytes. This is because of: (1) their peculiarsize and shape which are distinctly different from those ofwhite blood cells (WBCs); (2) the presence of molecularmarkers; and (3) the absence of a proliferative nature. Theseproperties make fNRBCs the prime candidate for the develop-ment of isolation-type microfluidic devices. These methodswill be discussed in later sections of this review.

3.2 Cell-free fetal DNA

The isolation and analysis of cell-free fetal DNA from thematernal plasma has shown tremendous potential for non-invasive prenatal diagnosis. In contrast to nucleated fetalcells, cell-free DNA is present abundantly in the maternalplasma constituting up to approximately 10% of maternalDNA.25,26 However, genetic information is muddled in thefragments of fetal DNA, which are typically only hundreds ofbase pairs in length, rendering them difficult to decipher andrequiring sophisticated molecular diagnostic techniques toretrieve fetal genetic information.27

Pioneering work by Lo et al.28,29 demonstrated that allelicratios of placental specific mRNA could be used to determinetrisomy 21. Honda et al. successfully showed detection of theY-chromosome using real time PCR and demonstrated sensitiv-ity of 100% as early as during the fifth gestational week.15

Cell-free fetal DNA can further facilitate the diagnosis of vari-ous chromosomal disorders such as aneuploidies, sex-linkeddisorders, beta-thalassemia, fetal rhesus D status and congeni-tal adrenal hyperplasia.30–34 Most of the commercial enterprisesin the field of NIPD are primarily based on cell-free fetal DNAanalysis and are discussed in detail in section 6 of this review.

The distinctive advantages and limitations of NIPD tech-niques, based on the isolation of cell-free fetal DNA andnucleated 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 Fetal cells

Advantages Higher abundance in maternal plasma (>10%)25 Pure, complete fetal DNA can be obtainedAssay for trisomies 21, 18 and 13 have been developed Whole genomic DNA amplification is possibleAutomated platform available Confirmation of fetal identity is possible in the 1st trimester

Disadvantages Fragmented nature of fetal DNA Extreme rarity of cells (1 cell ml−1 of maternal blood)35

High cost assay with lower throughput Automation is challengingSpecific assay needed to detect aneuploidy and genetic disorders Placental mosaicism within trophoblastic fetal cells17


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3.3 Etiology of disorders in NIPD

The presence of fetal abnormalities manifests itself inchanges in the number of fetal cells or amount of cell-freefetal DNA in the peripheral maternal blood when comparedto normal pregnancies. The usual count of fNRBCs detectedin the first trimester of normal pregnancy is reported to be upto two cells per milliliter of whole blood.35,36 The total num-ber of fNRBCs detected during aneuploidic pregnancies wasreported to be higher than the number of fNRBCs detectedduring normal pregnancies.37,38 Elevated fetal DNA levels inthe plasma are the basis for the diagnostic technique of cell-free fetal DNA analysis in cases of placental pathologies.39

Such etiologic changes have been observed quantitativelyin the following abnormalities for nucleated fetal cells andcell-free fetal DNA respectively. Polyhydramnios is the condi-tion where the amniotic fluid index is more than twice thestandard deviation above the mean when measured in thelate second to third trimesters. The cause of polyhydramnioscould be due to various maternal and fetal conditions likediabetes mellitus, congenital anomalies, iso-immunization,multiple gestation and placental abnormalities.40 Zhong et al.reported an excessive increase in the population of NRBCs inpolyhydramnios pregnancies at 34 weeks of gestation. Insuch cases, NRBC counts (~230), of both the mother and thefetus, were found to be higher than the counts in normalpregnancies (~5). This phenomenon was explained by theauthors as the escaping of large numbers of fNRBCs throughvilli to maternal uterine veins owing to the increase in pres-sure on the placenta during polyhydramnios. Other etiologicexamples are pre-eclampsia (PE) and intrauterine growthrestriction (IUGR). These have been responsible for fetal andmaternal mortality. The diagnostic biochemical markers cor-relating with the inflammatory response and endothelial dys-function for PE can only be detected at the end of the secondtrimester, thus making it difficult to introduce interventionto prevent the disease. On the other hand, the correlation ofPE and IUGR with the elevated levels of cell-free fetal DNA asearly as 11–14 weeks of pregnancy was evident in the studyby Illanes et al.41

The clearance of fetal DNA through renal pathways pro-vides an opportunity to perform NIPD using maternal urinesamples. However, past reports are inconclusive in determin-ing its feasibility in early diagnosis. Some recent reports42

have noted the difficulty in detecting cffDNA in maternalurine due to contamination arising from traces of spermDNA (e.g. occurrence of male DNA in cases of pregnancies

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with female foetuses) and quantities of cffDNA being lowerthan the detection limits.

4. Conventional approaches to NIPD

Conventional means of isolating fNRBCs can be classifiedinto 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 incombination. Density- and size-based methods for separatingcells are well-established. The most commonly used tech-niques are density gradient centrifugation (DGC)43 and size-based filtration. Fetal cell researchers have used centrifuga-tion on samples to reduce the amount of mature red bloodcells (RBCs) and to enrich the mononuclear cells in order toidentify their morphological characteristics. The mononuclearcells are characterized by immunocytochemical staining,44

May–Giemsa staining45 or the Pappenheim method.35 How-ever, centrifugation methods pose a potential risk of infectionto handlers due to aerosol generation, cause cell damage andresult in an estimated cell loss of 30 to 50%.43 The techniqueis laborious, requires accurate balancing of sample tubes andis difficult to integrate into continuous flow systems. A filtra-tion technique called “Isolation by Size of Epithelial TumorCells” (ISET) to isolate fetal trophoblasts was proposed byVona et al.46 It was able to successfully separate trophoblastsfor FISH as well as for DNA extraction and subsequent molec-ular analyses such as PCR. However, this method is not appli-cable as a standard NIPD technique due to the placentalmosaic nature of trophoblast cells.

Optical-based separation techniques include fluorescence-activated cell sorting (FACS) and laser micro-dissection.Researchers have shown that FACS can be used to identify fetalcells by relying on the detection of specific cell surface antigens,such as CD71 (transferrin receptor), CD36 (thrombospondinreceptor), and GPA (glycophorin A).47 Surface markers such asCD45, CD4, CD32, and CD19 can be used to remove unwantedcells such as WBCs.48,49 However, FACS set-ups usually consistof a laser source, optical components for light collection anddetection, as well as fluidic and electrical components whichaltogether make the technique capital-intensive and bulky.FACS-based sorting techniques are tedious and require well-trained personnel for operation.

Magnetic-based separation techniques can be appliedin either of two ways: (a) coating paramagnetic or ferromag-netic particles with cell-targeting molecules to achieve cell




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separation; or (b) using the intrinsic magnetic moment oftarget molecules such as deoxygenated RBCs to achieve this.Magnetic-activated cell sorting (MACS) uses specific super-paramagnetic bead-coated antibodies to coat target cells. Astrong magnet is then applied to immobilize bead-coatedcells while unlabeled cells are washed away. The immobilizedcells can then be collected by removing the magnet. MACS isreported to have poorer yield and purity compared to FACSand can lead to sample contamination and cell damage dueto shear forces. However, applying Ficoll gradient purificationto the samples before conducting MACS can improve theselectivity for NRBCs.50

Adhesion-based separation was performed by Kitagawa et al.51

using glass slides coated with a galactose-containing polymerand soybean agglutinin (SBA), a galactose-specific lectin,bound to the polymer surface. As galactose molecules arehighly expressed on the surface of erythroid precursor cells,NRBCs are enriched by their absorption to the slides whenthey pass by the surface and are captured by the SBA.However, screening the slides is labor-intensive and samplepurity is not high due to high amounts of contaminatingnon-nucleated RBCs.

Conventional processes to isolate fetal cells are laborious,time-consuming and difficult to automate. These problemscould be overcome using microfluidic technology.

5. Microfluidics in NIPD5.1 Microfluidic approaches using cell-free fetal DNA

PCR reactions have been explored in microfluidic systemsin detail. These have been developed in different materialslike 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 mainadvantages of microfluidics-based PCR devices.55 With therecent emergence of droplet microfluidic devices, the capabil-ity of performing thousands of compartmentalized, parallelPCR reactions in individual droplets has been imparted tomicrosystems.56 In digital PCR, DNA is quantified by countingthe instances of amplification of each copy of DNA. The PCRmixture along with the DNA template is diluted and distrib-uted within numerous compartments so that every compart-ment has less than one copy of DNA template and PCRproducts are detected by a fluorescence signal. Quantificationis achieved by correlating the number of DNA templates tothe number of compartments in which the signal is detected.This can be further used to detect copy number variationsor single nucleotide polymorphisms (SNPs) in the DNAsequence. The automation of digital PCR within microfluidicdevices has been explored in the past by Quake's group andhas been made commercially available by Fluidigm®.57 Chip-based digital PCR is also offered by Life Technologies in theform of the OpenArray™ platform.58 This consists of micro-fabricated through-holes which utilize hydrophobic coatings


to immobilize numerous droplets. Each array is capable ofcarrying out up to 36 000 parallel reactions. Another interest-ing microfluidic digital PCR approach, named SlipChip,59 wasproposed by Ismagilov's group. This system consists of glassplates with alignable microfabricated features for compart-mentalization of reactions isolated by oil-filled channels. Thesystem was reported to have good sensitivity and dynamicrange due to its multivolume approach.

Quake and co-workers showed the non-invasive diagnosisof fetal aneuploidy of trisomies 21, 18 and 13 using micro-fluidic digital PCR, as early as after 14 gestational weeks.60

The length of fetal DNA fragments (~169 bp, Fig. 2A) wasfound to be shorter than that of the maternal DNA (>250 bp),which is in agreement with previous reports.61 The authorswere able to successfully identify 9 cases of Down syndrome,2 cases of Edward syndrome and a case of Patau Syndromefrom a cohort of 18 patients. Interestingly, the findings of thiswork revealed that the majority of free DNA fragmentsobtained from the plasma shared features of nucleosomalDNA, further pointing out that the quantity of a particularlocus may not be representative of the quantity of the entirechromosome. Fan et al. showed the successful identificationof aneuploidies such as trisomy 21, trisomy 18, and trisomy13 in ongoing pregnancies with comparable accuracy usingthe same microfluidic digital PCR platform.62 However, thesamples were obtained by amniocentesis and CVS duringpregnancy. This in turn shows that microfluidic digital PCRcan be used either on its own as a technique to detect fetalaneuploidy non-invasively or integrated with current invasivediagnostic practices.

Whale et al.63 recently found that the microfluidic digitalPCR has better sensitivity than conventional quantitative PCRin detecting copy number variation (CNV), which is thechange in the genomic DNA of an individual leading to anabnormal number of copies of cffDNA. Additionally, theauthors pointed out that digital microfluidic PCR does notsuffer from the issues of technical variability in quantitativePCR, commonly observed between different laboratories.Dennis Lo and co-workers64 also demonstrated that micro-fluidic digital PCR has the least quantitative bias for the mea-surement of DNA fragments, higher precision and higherclinical sensitivity compared with conventional non-digitalreal-time PCR. The authors also reported that ZFY/ZFX assaysperformed on the microfluidic digital PCR platform (Fig. 2B)showed that the median fractional concentration of fetal DNAin the maternal plasma was two times more than previouslyreported during all three trimesters of pregnancy. Theserecent reports show the significant improvements that can bebrought to the field of NIPD using microfluidic digital PCR.

Tsui et al. demonstrated the application of microfluidicdigital PCR to the non-invasive detection of haemophilia byanalysis of maternal plasma.65 A pregnant haemophilic motherhas a 25% chance of carrying an affected male fetus. On theother hand 3–4% of fetuses suffer from cranial bleedingduring labor and pregnancy. The authors suggested a non-invasive 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, microfluidicdigital PCR offers higher sensitivity than the conventional RT-PCR.64 (Reproduced with permission from ref. 60 and ref. 64).


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carrier pregnancies, and were able to successfully identify 12clinical samples to be of a heterozygous nature for the causa-tive mutations. The quantity of fetal DNA was determinedusing the ZFY/X assay and found to be less than 10% of thetotal concentration of cell-free DNA. Using the microfluidicdigital PCR platform, Barrett et al.66 detected sickle cell anae-mia in 82% of male fetuses and 75% of female fetuses. Theyanalyzed the dosage of the variant encoding hemoglobin S(mutant) relative to that of hemoglobin A (wildtype) usingcffDNA from maternal plasma samples.

In addition to the microfluidic PCR platforms, Hahn andco-researchers67 had developed a dedicated microsystem topre-concentrate and separate fetal DNA from maternal plasmasamples. The platform was realised in PMMA and consistedof a polyethylene terephthalate membrane for electrokinetictrapping of cffDNA. The authors used a field-amplified samplestacking technique to concentrate the samples and were ableto concentrate 80 μl of the input sample into a final volume of2 μl, with the desired fraction of nucleic acids of targetedlength. The microsystem was eventually validated by separat-ing the DNA fragments (<500 bp) from maternal plasma andprobing them using real time PCR targeting the 107 bp SRY(sex determining region) sequence. Another microfluidic sam-ple preparation approach proposed by the Desmulliez group68

to isolate plasma from whole blood was based on theZweifach–Fung effect. Although the approach does not involveisolation of cffDNA, it can be easily extended to includecffDNA applications. In this particular work,68 the authorswere able to amplify the sequence of the circulating referencegene (GAPDH) without an additional step of purification.

Such platforms can replace the upstream manual steps ofsample preparation before digital microfluidic PCR. The inte-gration of these platforms has benefits, including: reductionin the potential risk of infection to an operator during centri-fugation; elimination of loss of smaller DNA fragments dur-ing sample processing; and the use of purification chemicalscan be avoided. Barrett et al.69 had described the importanceof sample preparation in retrieving cffDNA from blood

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samples for microfluidic digital PCR. The authors noticed asignificant increase in the total amount of copy number varia-tion of cell-free DNA (combined maternal and fetal DNA) perml of plasma. However, no increase in the CNV of cffDNA wasdetected. The increase in the total cell-free DNA was attributedto an increase in long maternal DNA strands released becauseof prolonged storage times. The authors suggested the use ofcell-stabilizing tubes or prompt preparation of plasma afterblood withdrawal as a measure to counter this effect. It can beinferred here that protocols for blood collection and samplepreparation can be of great significance for cffDNA methodol-ogies. The point-of-care nature of microfluidic devices canallow users to prepare the plasma promptly at a patient site inorder to nullify the effects of storage.

5.2 Microfluidic approaches to isolate fetal nucleated cells

Microfluidic attempts to isolate fNRBCs are mostly size exclu-sion-based. These will be discussed in detail in this section.

The work published by Huang et al.70 accomplished suc-cessful isolation of fNRBCs from maternal blood. The teamdeveloped a high-throughput and highly efficient microfluidicdevice for isolating rare NRBCs from maternal blood, inwhich 99.99% of non-target RBCs and WBCs were removed.They also successfully identified NRBCs from 58/58 sampleswith a mean of 37.44 NRBC mL−1 (range 0.37–274.36 NRBC mL−1).A modular approach was taken to first remove the RBCs fromthe whole blood and to leave nucleated cells behind, includ-ing WBCs and NRBCs. This was done using “Deterministiclateral displacement (DLD)”,71 a technique which exploitsthe hydrodynamic size differences between nucleated andnon-nucleated cells. The DLD principle uses the mechanismof laminar flow passing an array of micropillars. The flow isdivided into several different streams by the array of micro-pillars. Particles or cells smaller than the stream-width travelundisturbed in the streamlines (see red trajectories inFig. 3A). Larger particles are pushed laterally by the pillarsinto an adjacent stream (green trajectories in Fig. 3A). In



Fig. 3 Microfluidics approaches for fetal cells isolation. (A) Separation of fNRBCs and WBCs from RBCs by deterministic lateral displacement byHuang et al.70 (B) Isolation of fNRBCs by PDMS membrane technique by Kumo et al.72 (C) Micropillar filter by Mohammed et al. for isolation offNRBCs.73 (D) Cross filter design by Lee et al.74 (E) On-chip magnetophoretic fetal cell isolation approach proposed by Kavanagh et al.75


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blood separation, nucleated cells such as WBCs and fNRBCsare pushed in the lateral direction repeatedly and are sepa-rated from the RBCs, platelets and plasma at the outlet. Thisprocess was carried out in the cell separation microchip(CSM) to remove most of the RBCs (Fig. 3A). The second stepwas to isolate fNRBCs, using their paramagnetic properties,from the WBCs by sample treatment with 50 mM sodiumnitrite followed by application of a magnetic field. This treat-ment converts hemoglobin into methemoglobin in fNRBCs,which are then trapped in the hemoglobin enrichment (HE)module, consisting of a magnetic column and a magneticfield. The cells were collected from the HE module and stainedusing the Wright/Giemsa method. Manual enumeration wasthen performed to differentiate between typical NRBCs, atypi-cal NRBCs with non-standard morphology, and non-NRBC.The combined system could process 5 to 20 mL of maternalblood in 2 to 6 hours and needs about one hour for the auto-mated scanning and manual enumeration per microscopeslide for post-processing. The microfluidic device effectivelyeliminated about 99.99% of RBCs in the first step whereas thestationary capture of NRBCs on a magnetic column during thesecond HE steps allowed depletion of WBCs at about 99.90 to99.99% efficiency. The system was capable of identifying casesof trisomy 21 and trisomy 18 in the 11th to 21st weeks of gesta-tion, and trisomy 13 after 16 weeks of gestation.

Kumo et al.72 developed a microfluidic technique to iso-late fNRBCs from maternal blood using a microgap of 1 μmcreated by a PDMS membrane in a microchannel (Fig. 3B).The membrane was actuated by pressure so that it can be inan ‘open’ or ‘closed’ state to trap fNRBCs, due to the rigidnature of their 4–5 μm nuclei. The proposed technique


required sample preparation steps for obtaining a buffy coatafter density gradient centrifugation and a post-filtration stepof May-Giemsa staining to identify the fNRBCs. In-depthwork is further required to complete and validate this work.The above-mentioned two techniques are able to recover cellsafter their isolation, which is important for downstreammolecular analysis.

Mohamed and coworkers73 proposed a microfluidic deviceto separate fNRBCs from maternal circulation based on dif-ferences in their size and deformation characteristics. Thedevice (Fig. 3C) consisted of microposts fabricated in PDMS,separated by distances varying from 15 to 2.5 μm. Spikedgoose red blood cells and cord blood fetal cells were usedseparately to perform the experiments. The experimentsshowed that model fNRBCs which range in size from 9 to12 μm could not pass through a channel as small as 2.5 μmwide and 5 μm deep. Interestingly fNRBCs from the cordblood could pass through the channel whereas the whiteblood cells, which ranged in sizes from 10 to 20 μm, couldnot be deformed and were retained in the upstream gaps. Inthis work, goose red blood cells were used as fNRBCs due totheir size which, at 12 μm, is similar to 10 to 8 μm-sizedfNRBCs. The authors reported the isolation of fNRBCs fromWBCs in cord blood based on cell size and deformation char-acteristics. However, the experiments could not illustrate thepresence of intact fNRBCs at the outlet of the device. Molecu-lar analysis of the eluate indeed showed the usefulness ofthis technique in enriching the sample by filtering out WBCsinside the microfluidic device. However, the sample process-ing steps such as DGC, dilution of cord blood samples andinability to handle larger samples make this method difficult

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Fig. 4 (A) Dielectrophoresis chip to isolate fNRBCs from sparse samples.76

(B) DEPArray™ technology from Silicon Biosystems.82 (Reproducedwith permission from Silicon Biosystems Inc.).


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to implement in clinical settings. The flow rate in this deviceis also very low, about 0.35 mL per hour, which will not bepractical for processing 10–20 mL whole blood samples inclinical practice.

Lee et al.74 presented a continuous microfluidic approachto separate fNRBCs from RBCs in model mixtures preparedusing different ratios of fNRBCs/RBCs. A cross-flow filterdevice (Fig. 3D) was fabricated in silicon with two inlets toinject the model mixtures and buffer solution. The deviceconsisted of microfiltering structures arranged in a particularorientation to filter fNRBCs into an adjacent stream of PBSbuffer and to allow RBCs to pass through the gaps betweenthe structures. The viability of fNRBCs was determined byepsilon-globin immunostaining to confirm the integrity ofcell morphology and cytoplasmic contents. This also provedthat hydrodynamic pressure generated at moderate flow ratesdid not adversely affect fNRBCs. The experiments conductedwere able to demonstrate 74% fNRBC recovery and 46.5%RBC depletion. Although this work is an interesting exampleof fNRBC isolation in microfluidic devices, the fNRBCs/RBCratios used (1 : 100) were not exact representations of physio-logical conditions (1 : 107). The maternal blood samples wouldeven typically contain fewer WBCs after WBC removal, whichmight pose clogging issues for processed blood or dilutedblood samples in the proposed device.

Kavanagh et al.75 proposed a multi-modular microfluidicapproach for isolation of fetal cells from whole blood. The firststage (Fig. 3E) involves separation of plasma and blood cellsusing a microfluidic channel designed on the basis of theZweifach–Fung effect. The subsequent module was designed toremove WBCs from the remaining blood cells based onimmunomagnetic principles. The final module removes RBCsusing their paramagnetic properties and fNRBCs are finally col-lected at the outlet. The approach, however, lacked sufficientbiological validation using spiked or clinical samples. Notice-ably, this was the first microfluidic attempt wherein authorstried a two-way approach to isolate fetal cells and retrievegenetic material from the blood plasma, from the same sample.

An attempt to study fetal cell properties using microfluidicdevices was presented by Xu et al.76 wherein dielectrophoresis(DEP) was used to trap the fNRBCs and to determine thecrossover frequency of fetal cells. DEP cell separation is possi-ble because different types of cell in blood respond differentlyto an electrical field, depending on the frequency applied(Fig. 4A). By adjusting the field frequency and amplitude, cellscan be manipulated. Xu et al. successfully demonstrated DEPin a microfluidic chip for cell isolation. However, the methodsuffered from electrode fouling and inconsistent accuracy.Such a method will require prior steps of sample preparationto enrich the sample and is not capable of high throughputhandling of samples. A platform based77 on the dielectrophoreticmanipulation of cells has been commercialised by Silicon Bio-systems using DEP pixel-based manipulation of rare cells,such as fetal cells (Fig. 4B and section 6).

The techniques above were developed to isolate fetalcells. However, diagnostic detection of trisomies within the

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microfluidic devices was not explored. Ho et al.78 developeda microfluidic FISH method which can be carried out within3 hours, as opposed to a total processing time of 24–48 hoursfor conventional FISH techniques. Even though clinical sam-ples were obtained using amniocentesis, the work success-fully demonstrated the following benefits of carrying outcomplete molecular diagnosis within the microenvironment:(1) economical use of reagents with a 20-fold decrease in theamount of FISH reagents needed; (2) localization of fetal cellswithin a confined area leading to easier visualization andimaging; and (3) reduced assay time. The new tests carriedout within the microfluidic device were able to reduce thecost of FISH analysis by almost half, with due considerationof the capital needed for fabricating the device.

6. Commercial entities in NIPD

The future market of NIPD was reported to be more than1 billion dollars79 and this necessitates focusing on some ofthe key commercial entities in this review. Most commercialentities use the cffDNA approach, with the exception ofKellBenx, Silicon Biosystems and CellScape.

KellBenx incorporation80 (USA) adopts a cell isolationapproach based on molecular affinity using the proprietarymonoclonal antibody, 4B9. 4B9 has an affinity for epitopesexpressed on the fNRBC surface. This approach81 requiresthe additional steps of density centrifugation to remove theplasma followed by the washing and resuspension of cells,




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which might increase fetal cell loss. Remarkably, the com-pany has reported a recovery of 60–120 fNRBCs ml−1 frommaternal blood in the first trimester, in contrast to priorattempts which reported 1–2 cells ml−1.

Silicon Biosystems82 in Bologna, Italy markets adielectrophoresis cage-based instrument for enriching sam-ples with a heterogeneous population of rare cells, such asfetal cells. In this system the difference in dielectrophoreticproperties of different cells is exploited to isolate fNRBCsfrom RBCs and WBCs.

The company has developed an automated platformnamed DEPArray™, which is capable of identifying, manipu-lating and sorting individual cells in samples containing 1 to100 000 cells. The automated instrumentation allows unprec-edented control over single cells but requires sample prepara-tion steps such as DGC and MACS, in addition to highercosts for installation.

CellScape Corp.83 in California is working on the isolationof fetal cells from maternal blood. CellScape has developed atest called ‘Clarity™ Prenatal Genetic Test’ which is availablefor investigative purposes. Clarity™ needs 16 ml of maternalblood, which is processed to remove background cells, enrichedby optical observation, and finally probed for fNRBCs. Thetest uses chromosomal microarray analysis (CMA) to reliablydetect a targeted set of genetic disorders. The test can identifytrisomies 13, 18 and 21, and will be expanded to detect cysticfibrosis and sickle cell anemia in the future.

Verinata Health Inc.85 (USA) has commercialised a cffDNA-based NIPD test called ‘Verifi® prenatal test’. This system iscapable of identifying aneuploidies such as trisomies 21, 18and 13 based on massively parallel DNA sequencing. In addi-tion, early this year Verinata announced the ability to detectmonosomy X or Turner syndrome, which is prevalent in preg-nancies positive for cystic hygroma in ultrasound analyses.In addition to the diagnosis of Turner syndrome, Verinata hassuccessfully diagnosed Klinefelter syndrome (XXY), Triple Xsyndrome (XXX) and XYY syndrome in patients. Verinata wasacquired by Illumina Inc. recently.

Sequenom Bioscience,88 from San Diego in the USA, is apioneering company that successfully commercialised NIPDtesting for detecting Down syndrome based on cffDNA analy-sis. Clinical trials have also demonstrated identification oftrisomies 18 and 13 with detection rates of 100% and 91.7%.These trials yielded false positive rates of 0.28% and 0.97%,respectively, with sample sizes as large as 1988 patients. Falsepositive rates as low as 0.1%, and with an overall detectionrate of 98.9% for all common aneuploidies, were alsoreported. The company markets the ‘MaterniT21 Plus’ test,capable of detecting relative amounts of chromosomes 21,18, 13 and Y material. Cell-free DNA is separated from mater-nal blood plasma, analyzed for autosomal and Y chromo-somal material, then converted into a genomic DNA librarybased on massively parallel DNA sequencing.

A company based in San Jose, USA, Ariosa Diagnostics,Inc.92 (formerly known as Aria Inc.) has commercialised‘Harmony™ Prenatal test’ to identify pregnancies with trisomies


21, 18 and 13. This test is based on direct DNA analysis(or targeted sequencing) of cffDNA within the maternalplasma obtained after at least 10 weeks of gestation. A detec-tion rate of more than 98% and a false positive rate of lessthan 0.1% were reported with over 6000 patients.

Natera Inc.96 from San Carlos, USA has launched its prena-tal diagnostic test ‘Panorama Prenatal Test’ to perform fetalpaternity testing and prenatal aneuploidy detection usingcffDNA analysis. PreNATUS (Prenatal Non-invasive AneuploidyTesting Using SNPs), a blinded clinical trial to validate theParental Support™ technology from Natera is in progress inmore than 20 clinical centres in the USA.

Apart from the above-mentioned major commercial enti-ties, the following companies are present on the NIPD land-scape too. Predominantly, these companies are relying oncffDNA methods with the exception of the Abnova platform.These commercial entities are listed here: Berry Genomics98

(BambniTest™) uses a massively parallel sequencing approachfor identification of aneuploidies; Abnova Corp. (FetoLumina™platform99) offers a platform based on isolation of fNRBCsfrom blood; Ravgen offers paternal, Down syndrome and sin-gle gene disorder testing based on cffDNA analysis;100 andNIPD genetics,101 Cyprus and BGI Health102 in China offeraneuploidy detection based on cffDNA analysis. A comparisonbetween these commercial entities is provided in Table 2.

We would like to discuss upcoming challenges that will befaced by these commercial entities. Mennuti et al.103 haveraised concerns about imperfections in the positive predictivevalue of cffDNA methods. The authors explained that theinherent nature of cffDNA tests yields false positive/negativeresults due to the requirement for a quantitative cut-off topredict the nature of pregnancy. Despite the very low falsepositive rates reported (<0.5%) for trisomy 21 and trisomy 18using NIPD tests, Mennuti et al. described eight verifiedcases of false positives after performing invasive prenataldiagnosis. The authors also recommend that validation stud-ies be extended to low-risk pregnancies for better under-standing of false positive rates and readjustment of the falsepositive value for the general population. A similar concernwas raised by Reiss and Cherry104 about the confusionbetween high specificity and high positive predictive value.In fact, many recent groups104–107 have chosen to addresscffDNA-based non-invasive prenatal diagnostics (NIPD) methodsas non-invasive prenatal testing (NIPT) methods because oftheir need of invasive procedures such as amniocentesis orCVS after getting positive results and the quantitative predic-tive form of test results. The American College of Obstetri-cians and Gynaecologists105 has advised that cffDNA-basedmethods should not be a part of routine lab assessment butshould be “an informed patient choice”105 after counselling.

Canick and researchers106 investigated the correlation offetal fraction with the screening performance for trisomy 21using NGS methods. The fetal fraction was found to varyfrom 3 to 30% (in 212 and 1484 cases of trisomy 21 andeuploidic conditions, respectively), and it was observed thatthe screening performance increases as the fetal fraction

Lab Chip, 2014, 14, 841–854 | 849


Table 2 Comparison between commercial entities in non-invasive prenatal diagnostics (NIPD)

ApproachDiagnosismethod Test Fetal conditions Sensitivity

Gestationalperiod of fetus Cost $

Turnaroundtime Reference

Sequeonom cffDNA Massivelyparallelsequencing

MaterniT21plus

13, 18, 21, sexchromosomesaneuploidies

92–99% 10 weeks 1700 (op) 8–10 days Ehrich et al.89

235 (cp) Bombardet al.90

2900 (ip) Palomakiet al.91

Natera cffDNA Singlenucleotidepolymorphism

Panoramaprenatal test

13, 18, 21, sexchromosomesaneuploidies

92–99% 9 weeks Unknown(op)

15 days Zimmermanet al.97

1495 (ip)Ariosa cffDNA Targeted

sequencingHarmonyprenatal test

13, 18, 21 80–99% 10 weeks 795 (op) 8–10 days Ashoor et al.93

Diagnostics 95 (cp) Norton et al.94

Nicolaideset al.95

KellBenxInc.

fNRBCs Standardmoleculardiagnosis

NA 13, 18, 21, X, Y NA 6–12 weeks NA NA 80,81

Verinata cffDNA Massivelyparallelsequencing

Verifi® 13, 18, 21, sexchromosomesaneuploidies

87–99% 10 weeks 295 (op) 8–10 days Sehnert et al.86

Health 200 (cp) Bianchi et al.87

1200 (ip)Siliconbiosystems


DEPArray™a NA NA NA NA NA Borgattiet al.77,82

Berrygenomics

cffDNA Massivelyparallelsequencing

BambniTest™ 13, 18, 21, sexchromosomesaneuploidies

100% 10 weeks 500 (op) 7 days 98

CellScapeInc.


Clarity™prenatal testb

13, 18, 21, X, Y NA 6 weeksonwards

NA NA Parikhet al.83,84

Abnova fNRBCs Standardmoleculardiagnosis

FetoLumina™c 13, 18, 21, X, Y NA 7 weeksonwards

NA NA 99

Abbreviations: op = out-of-pocket, cp = co-pay with insurance coverage, ip = billed to insuring company,108 NA = not available or applicable,$ = US dollars.a The platform can be used to isolate fNRBCs which can be studied by standard technique such as FISH, CGH or NGS.b The test is available only for investigational purposes at the current moment. c Limited information available.


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increases. Interestingly, the authors identified a correlationbetween maternal weight and fetal fraction, wherein low fetalfractions corresponded to high maternal weight and anincrease in false negatives. The authors also highlighted thecasual effects of placental mosaicism on the fetal fraction,resulting in its inaccurate determination.

In view of the nature of massively parallel sequencing(MPS) and targeted sequencing, cffDNA methods necessarilyincur costs in terms of time and money.107 MPS approachestake longer processing durations and are more expensive buthave higher accuracy. A shift towards the most cost effectiveapproach was anticipated by Boon et al.107 The authors alsocategorically highlighted the higher price of NIPD/NIPT tech-niques, stressing that this could reflect commercial pricesand not cost prices. The authors predicted an easing of theprices in the near future, with Agarwal et al.108 pointing outthat insurance companies have recently covered the cost ofsome commercial tests. The ultimate challenge for existingNIPD tests is to be accurate enough to be independently

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conclusive27 without the need for additional invasive analyses.They also have to be cheaper than the invasive alternatives.

7. Future scope7.1 Challenges in the field of NIPD

In this review paper, we have given a brief outline of currentstandards of prenatal diagnostics and NIPD. Readers will beable to appreciate the fact that microfluidic approaches haveinfluenced NIPD significantly and will continue to play a keyrole in the near future. Microfluidic digital PCR has been pro-viding an accurate and highly sensitive alternative to the cur-rent technique of qPCR to replace standard droplet-basedPCR techniques in specific applications. We anticipate thatmicrofluidic digital PCR techniques will shape the future ofNIPD and will have a significant impact in the clinical set-ting. Although, the cffDNA-based approach has been adoptedby clinicians and marketed by commercial entities, the goalof prenatal diagnosis could be more readily achieved with the




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non-invasive retrieval of complete and intact fetal genomesfor molecular analysis. From that perspective, isolating fetalcells from maternal blood still has an immense potential.

Hereby, we list six major challenges that should be consid-ered by the microfluidic community.

Large sample volumes. The usual volume of blood withdrawnfrom pregnant patients is usually 20 ml for isolating fetalcells, whereas the amount of blood collected for cffDNAanalysis can vary from 2–4 ml. Microfluidic devices are oftenhighlighted for their ability to handle smaller sample volumesbut would probably need a longer process for maternal bloodsamples. This can be decisive when isolating fNRBCs fromblood. Alternatively, a microfluidic approach can be introducedafter off-chip processing of samples, such as for obtaining abuffy coat after density gradient centrifugation. However, theloss of rare cells during sample processing has to be weighedagainst the benefits of a high-throughput approach in order tocome up with the ideal method.

Fetal cell shape and number. The problem of fetal cellsis indeed more complex than that of circulating tumorcells (CTCs)109 which have a peculiar size, rigid morphologyand relatively higher concentration in blood. Lab-on-a-chipapproaches for isolating fNRBCs – regardless of theirinnovative nature – have so far been limited to the use of sizeexclusion. It has to be understood that isolation of cells isinsufficient. What is also essential is their recovery, intact,from the device for downstream analyses. A concerted effortis necessary to develop microfluidic techniques which canisolate and recover cells. One possibility could be a techniquebased on inertial principles using Dean's flow to isolatefNRBCs. Optical-, adhesion-, antibody- and magnetic-basedapproaches still have the potential to be implemented inminiaturised devices. Recent advances in microfluidics suchas FACS-on-chip,110 relying on optical and adhesion princi-ples, can also be applied to isolate fetal cells.

Clinical validation of microfluidic systems. Isolating fetalcells (especially fNRBCs) from maternal blood was addressedsolely by Huang et al. Other efforts were limited to the use ofmodel mixtures or model cells, which are a far cry from realclinical samples and an over-simplification of the problem.Current microfluidic attempts at isolating fetal cells need tobe rigorously validated using clinical samples. The samplepool also has to be diverse, including patients with or with-out high-risk pregnancy, to avoid sample bias.

Automation. Undoubtedly, the microfluidic approach withsingle or multi-modular components requires certain opera-tional skills which are uncommon in clinical settings. Theautomation of microfluidic approaches will eliminate thislimitation and will impart the platform with clinical applica-bility. This would further help to eliminate user-bias which iscrucial for sound clinical validation.

Integration of sample preparation. This is one of the mostchallenging processes to be implemented upstream of currentcell-free DNA techniques using digital microfluidic PCR. Thesample preparation techniques mentioned in this review willbe useful for improving DNA recovery.


Physiological understanding of placenta and cell transfer.In addition to prenatal diagnosis (isolation of fetal cellsand cffDNA), microfluidics can play a significant role inthe fundamental understanding of placental development,pregnancy disorders, and why and how these cells appear in thematernal circulation.111–113 The mechanism of cell exchangeacross the placenta is still unclear113 and could be addressedusing microfluidic networks mimicking placental anatomy.Such studies would help to elucidate the links betweenphysiological conditions and their physical manifestations inconditions such as polyhydramnios or pre-eclampsia.

Lastly, this review would not be complete without under-standing the ethical aspects of NIPD and how it wouldimpact society. This is discussed in the last section.

7.2 Ethical aspects

A careful consideration of ethical issues will decide the futureof prenatal diagnostics. It would also affect the perception ofNIPD by various political, religious and social groups andhave an impact on the future of forthcoming generations.

The advantages of prenatal diagnostics bring along thepossibilities of adverse effects that have been witnessed previ-ously in Asian countries like India, China, Nepal and Vietnamleading to gender biased termination of pregnancies and seri-ous declining of the sex-ratio in some of these countries.114 Arecent review by Benn et al.115 highlighted three major con-cerns: (1) development of a trivial outlook towards pregnancytermination due to the use of NIPD might increase the fre-quency of termination; (2) this could also change the attitudeof society towards individuals suffering from Down syndromeand similar disorders, and could lead to discriminationagainst such individuals and their families in social and eco-nomic circumstances such as medical care; (3) the lack of pre-NIPD counseling for pregnant women. These concerns evokea strong need for ethical consideration from the medical com-munity, which include regular counseling during maternalcare, legislations from governmental bodies to protect theinterests of individuals and ultimately spreading awarenessabout NIPD among the public. Schmitz et al.116 pointed outthat this lack of sufficient counseling could also adverselyaffect post-NIPD decision making and could hamper thereproductive autonomy of the pregnant woman. However,Ravitsky117 countered by arguing that NIPD would, in fact,strengthen the reproductive autonomy of the female by miti-gating the risk of losing a healthy fetus due to invasive proce-dures. This would, in turn, shape NIPD counseling.

Abortion is itself a highly contested issue and has alreadybeen embroiled in the religious and political dogmas, whichwould make it difficult to look at this issue in a rational way.Termination of pregnancy based on NIPD could become com-monplace and could lead to trivialization of abortion, whereeach case of NIPD testing enforces impending probability ofpregnancy termination on the unborn baby. This eventuallybrings up the controversial issue of eugenics and the necessityto predetermine the thresholds of “normalcy” for pregnancy.

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The definition of normalcy of pregnancy would furtherdemand the ethical understanding of how and who will decidethe normalcy of the fetus, and whether our understanding offetal development is sound enough to make such intricate deci-sions; and as provocatively put forth by the authors118 to drawa distinguishing line between an unborn fetus and a chattel.On the other side of this ethical dilemma lie the benefits ofearly identification and termination of abnormal pregnanciesfor those who consider the abortion of fetus to be ethically andmorally less problematic in its early stage.

Acknowledgements

Authors would like to thank Dr Angela N. Barrett for insight-ful discussions, and Dr Darren C. W. Tan for proofreadingthis manuscript.

Notes and references

1 M. V. Steele and V. R. Berg, Lancet, 1966, 1, 383.
2 D. A. Driscoll and S. Gross, N. Engl. J. Med., 2009, 360,
2556–2562.3 T. Hassold, M. Abruzzo, K. Adkins, D. Griffin, M. Merrill,

E. Millie, D. Saker, J. Shen and M. Zaragoza, Environ. Mol.Mutagen., 1996, 28, 167–175.

4 E. A. Papageorgiou and P. C. Patsalis, Genome Med., 2012,
4, 46–2012.
5 D. M. Kavanagh, M. Kersaudy-Kerhoas, R. S. Dhariwal and
M. P. Y. Desmulliez, J. Chromatogr., B: Anal. Technol.Biomed. Life Sci., 2010, 878, 1905–1911.
6 J. L. Simpson, Best Pract. Res., Clin. Obstet. Gynaecol., 2012,
26, 625–638.
7 J. L. Simpson, N. Engl. J. Med., 2005, 353, 2068–2070.
8 U. Elchalal, I. B. Shachar, D. Peleg and J. G. Schenker,
Fetal Diagn. Ther., 2004, 19, 195–198.9 ACOG Practice Bulletin No. 27. Clinical Management

guidelines for obstetrician-gynaecologists. Prenatal diagnosisof fetal chromosomal abnormalities, Obstet. Gynecol., 2001,97, 12.

10 Amniocentesis and chorionic villus sampling (Green-top 8),
Royal College of Obstetricians and Gynaecologists, 2010,pp. 1–13.
11 E. Pergament, J. D. Schulman, K. Copeland, B. Fine,
N. A. Ginsberg, M. C. Frederiksen and R. J. Carpenter,Prenatal Diagn., 1992, 12, 377–384.
12 H. W. Lawson, A. Frye, H. K. Atrash, J. C. Smith,
H. B. Shulman and M. Ramick, Am. J. Obstet. Gynecol.,1994, 171, 1365–1372.
13 K. H. Nicolaides, Am. J. Obstet. Gynecol., 2004, 191, 45–67.
14 F. D. Malone, H. Cuckle, R. H. Ball, D. A. Nyberg,
C. H. Comstock, G. Saade, R. L. Berkowitz, S. J. Gross,L. Dugoff and S. D. Craigo, Am. J. Obstet. Gynecol., 2005, 193.

15 H. Honda, N. Miharu, Y. Ohashi, O. Samura, M. Kinutani,
T. Hara and K. Ohama, Hum. Genet., 2002, 110, 75–79.
16 H. Mouawia, A. Saker, J. Jais, A. Benachi, L. Bussieres,
B. Lacour, J. Bonnefont, R. Frydman, J. Simpson and
852 | Lab Chip, 2014, 14, 841–854

P. Paterlini-Brechot, Reprod. BioMed. Online, 2012, 25,508–520.

17 S. Hahn, R. Sant and W. Holzgreve, Mol. Hum. Reprod.,
1998, 4, 515–521.
18 C. Beroud, M. Karliova, J. P. Bonnefont, A. Benachi,
A. Munnich, Y. Dumez, B. Lacour and P. Paterlini-Brachot,Lancet, 2003, 361, 1013–1014.
19 A. Saker, A. Benachi, J. P. Bonnefont, A. Munnich,
Y. Dumez, B. Lacour and P. Paterlini-Brechot, PrenatalDiagn., 2006, 26, 906–916.
20 M. Choolani, A. P. Mahyuddin and S. Hahn, Best Pract.
Res., Clin. Obstet. Gynaecol., 2012, 26, 655–667.
21 D. W. Bianchi, G. K. Zickwolf, M. C. Yih, A. F. Flint,
O. H. Geifman, M. S. Erikson and J. M. Williams, PrenatalDiagn., 1993, 13, 293–300.
22 C. Troeger, W. Holzgreve and S. Hahn, Prenatal Diagn.,
1999, 19, 521–526.
23 D. W. Bianchi, G. K. Zickwolf, G. J. Weil, S. Sylvester and
M. A. Demaria, Proc. Natl. Acad. Sci. U. S. A., 1996, 93,705–708.
24 D. W. Bianchi, Br. J. Haematol., 1999, 105, 574–583.
25 Y. M. D. Lo, M. S. Tein, T. K. Lau, C. J. Haines, T. N. Leung,
P. M. Poon, J. S. Wainscoat, P. J. Johnson, A. M. Chang andN. M. Hjelm, Am. J. Hum. Genet., 1998, 62, 768–775.

26 A. G. Edlow and D. W. Bianchi, Biochim. Biophys. Acta,
1822, 2012, 1970–1980.
27 S. Hahn, O Lapaire, S. Tercanli, V. Kolla and I. Hösli,
Expert Rev. Mol. Med., 2011, 13, e 16.
28 Y. M. D. Lo, N. Corbetta, P. F. Chamberlain, V. Rai,
I. L. Sargent, C. W. Redman and J. S. Wainscoat, Lancet,1997, 350, 485–487.
29 Y. M. D. Lo, N. B. Y. Tsui, R. W. K. Chiu, T. K. Lau,
T. N. Leung, M. M. S. Heung, A. Gerovassili, Y. Jin,K. H. Nicolaides, C. R. Cantor and C. Ding, Nat. Med., 2007,13, 218–223.
30 C. P. Chen, S. R. Chern and W. Wang, Clin. Chem., 2001,
47, 937–939.
31 J. M. Costa, A. Benachi and E. Gautier, N. Engl. J. Med.,
2002, 346, 1502.
32 K. M. Finning, P. G. Martin, P. W. Soothill and N. D. Avent,
Transition Met. Chem., 2002, 42, 1079–1085.
33 R. W. K. Chiu, T. K. Lau, T. N. Leung, K. C. K. Chow,
D. H. K. Chui and Y. M. Dennis Lo, Lancet, 2002, 360,998–1000.
34 R. W. K. Chiu, T. K. Lau, P. T. Cheung, Z. Q. Gong,
T. N. Leung and Y. M. D. Lo, Clin. Chem., 2002, 48, 778–780.
35 H. Takabayashi, S. Kuwabara, T. Ukita, K. Ikawa, K. Yamafuji
and T. Igarashi, Prenatal Diagn., 1995, 15, 74–77.
36 D. W. Bianchi, J. M. Williams, L. M. Sullivan, F. W. Hanson,
K. W. Klinger and A. P. Shuber, Am. J. Hum. Genet., 1997, 61,822–829.
37 L. Zhang, Y. Wang and A. H. Liao, Prenatal Diagn., 2008,
28, 1160–1166.
38 Y. M. D. Lo, T. K. Lau, J. Zhang, T. N. Leung, A. M. Z. Chang,
N. M. Hjelm, R. S. Elmes and D. W. Bianchi, Clin. Chem.,1999, 45, 1747–1751.



Publ

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ity L

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ry o

n 30

/09/

2014

11:

59:5

6.

View Article Online

39 S. Hahn, B. Huppertz and W. Holzgreve, Placenta, 2005, 26,
515–526.
40 X. Y. Zhong, W. Holzgreve, J. C. Li, K. Aydinli and S. Hahn,
Prenatal Diagn., 2000, 20, 838–841.
41 S. Illanes, M. Parra, R. Serra, K. Pino, H. Figueroa-Diesel,
C. Romero, J. A. Arraztoa, L. Michea and P. W. Soothill,Prenatal Diagn., 2009, 29, 1118–1122.
42 S. Majer, M. Bauer, E. Magnet, A. Strele, E. Giegerl, M. Eder,
U. Lang and B. Pertl, Prenatal Diagn., 2007, 27, 1219–1223.
43 G. Sitar, L. Manenti, A. Farina, V. Lanati, P. Mascheretti,
A. Forabosco, L. Montanari and E. Ascari, Haematologica,1997, 82, 5–10.
44 J. C. Oosterwijk, W. E. Mesker, M. C. M. Ouwerkerk,
C. F. H. M. Knepfle, M. J. M. Van der Burg, C. C. Wiesmeijer,G. C. Beverstock, M. Losekoot, L. F. Bernini, G. J. B. VanOmmen, J. J. P. Van de Kamp, H. H. H. Kanhai andH. J. Tanke, Early Hum. Dev., 1996, 47, S95–S97.
45 R. Al-Mufti, C. Lees, G. Albaiges, H. Hambley and
K. H. Nicolaides, Hum. Reprod., 2000, 15, 218–221.
46 G. Vona, C. Beroud, A. Benachi, A. Quenette, J. P. Bonnefont,
S. Romana, Y. Dumez, B. Lacour and P. Paterlini-Brechot,Am. J. Pathol., 2002, 160, 51–58.
47 D. W. Bianchi, G. K. Zickwolf, M. C. Yih, A. F. Flint,
O. H. Geifman, M. S. Erikson and J. M. Williams, PrenatalDiagn., 1993, 13, 293–300.
48 D. E. Lewis, W. Schober, S. Murrell, D. Nguyen, J. Scott,
J. Boinoff, J. L. Simpson, F. Z. Bischoff and S. Elias,Cytometry, 1996, 23, 218–227.
49 J. L. Simpson and S. Elias, JAMA, J. Am. Med. Assoc., 1993,
270, 2357–2361.
50 I. Safarik and M. Safarikova, J. Chromatogr. B: Biomed. Sci.
Appl., 1999, 722, 33–53.
51 M. Kitagawa, K. Sugiura, H. Omi, Y. Akiyama, K. Kanayama,
M. Shinya, T. Tanaka, H. Yura and H. Sago, Prenatal Diagn.,2002, 22, 17–21.
52 C. J. Easley, J. M. Karlinsey, J. M. Bienvenue, L. A. Legendre,
M. G. Roper, S. H. Feldman, M. A. Hughes, E. L. Hewlett,T. J. Merkel, J. P. Ferrance and J. P. Landers, Proc. Natl.Acad. Sci. U. S. A., 2006, 103, 19272–19277.
53 J. Khandurina, T. E. McKnight, S. C. Jacobson, L. C. Waters,
R. S. Foote and J. M. Ramsey, Anal. Chem., 2000, 72,2995–3000.
54 C. Zhang and D. Xing, Nucleic Acids Res., 2007, 35,
4223–4237.
55 J. Yang, Y. Liu, C. B. Rauch, R. L. Stevens, R. H. Liu,
R. Lenigk and P. Grodzinski, Miniaturisation for Chemistryand Biology, Lab Chip, 2002, 2, 179–187.
56 B. G. Zimmermann, S. Grill, W. Holzgreve, Y. Z. Xiao,
L. G. Jackson and S. Hahn, Prenatal Diagn., 2008, 28,1087–1093.
57 M. Vaninsberghe, K. A. Heyries and C. L. Hansen, Expert
Rev. Mol. Diagn., 2012, 12, 111–114.
58 LifeTechnologies™, www.lifetechnologies.com.
59 F. Shen, W. Du, J. E. Kreutz, A. Fok and R. F. Ismagilov,
Miniaturisation for Chemistry and Biology, Lab Chip, 2010,10, 2666–2672.


60 H. C. Fan, Y. J. Blumenfeld, U. Chitkara, L. Hudgins and
S. R. Quake, Proc. Natl. Acad. Sci. U. S. A., 2008, 105,16266–16271.
61 K. C. A. Chan, J. Zhang, A. B. Y. Hui, N. Wong, T. K. Lau,
T. N. Leung, K. W. Lo, D. W. S. Huang and Y. M. D. Lo,Clin. Chem., 2004, 50, 88–92.
62 H. C. Fan, Y. J. Blumenfeld, Y. Y. El-Sayed, J. Chueh and
S. R. Quake, Am. J. Obstet. Gynecol., 2009, 200, 543.e541–543.e547.
63 A. S. Whale, J. F. Huggett, S. Cowen, V. Speirs, J. Shaw,
S. Ellison, C. A. Foy and D. J. Scott, Nucleic Acids Res., 2012,40, 82–89.
64 F. M. F. Lun, R. W. K. Chiu, K. C. A. Chan, Y. L. Tak,
K. L. Tze and Y. M. D. Lo, Clin. Chem., 2008, 54, 1664–1672.
65 N. B. Y. Tsui, R. A. Kadir, K. C. A. Chan, C. Chi, G. Mellars,
E. G. Tuddenham, T. Y. Leung, T. K. Lau, R. W. K. Chiuand Y. M. D. Lo, Blood, 2011, 117, 3684–3691.
66 A. N. Barrett, T. C. R. McDonnell, K. C. A. Chan and
L. S. Chitty, Clin. Chem., 2012, 58, 1026–1032.
67 T. Hahn, K. S. Drese and C. K. O'Sullivan, Clin. Chem.,
2009, 55, 2144–2152.
68 M. Kersaudy-Kerhoas, D. Kavanagh, R. Dhariwal, C. Campbell
and M. P. Y. Desmulliez, Lab Chip, 2010, 10, 1587–1595.
69 A. N. Barrett, B. G. Zimmermann, D. Wang, A. Holloway
and L. S. Chitty, PLoS One, 2011, 6, e25202.
70 R. Huang, T. A. Barber, M. A. Schmidt, R. G. Tompkins,
M. Toner, D. W. Bianchi, R. Kapur and W. L. Flejter,Prenatal Diagn., 2008, 28, 892–899.
71 L. R. Huang, E. C. Cox, R. H. Austin and J. C. Sturm,
Science, 2004, 304, 987–990.
72 T. Kumo, Y. Tomizawa, M. Kita, H. Takabayashi, E. Tamiya
and Y. Takamura, 14th International Conference onMiniaturized Systems for Chemistry and Life Sciences,Groningen, The Netherlands, pp. 1583–1585.
73 H. Mohamed, J. N. Turner and M. Caggana, J. Chromatogr.,
A, 2007, 1162, 187–192.
74 D. Lee, P. Sukumar, A. Mahyuddin, M. Choolani and G. Xu,
J. Chromatogr., A, 2010, 1217, 1862–1866.
75 D. M. Kavanagh, M. Kersaudy-Kerhoas, S. K. Pavuluri,
R. S. Dhariwal and M. P. Y. Desmulliez, 2nd Micro andNano Flows Conference UK, 2009, pp. 1–6.
76 G. Xu, M. B. Chan, C. Yang, P. Sukumar, M. Choolani and
J. Y. Ying, J. Phys. Conf. Ser., 2006, 34, 1106–1111.
77 M. Borgatti, N. Bianchi, I. Mancini, G. Feriotto and
R. Gambari, Int. J. Mol. Med., 2008, 21, 3–12.
78 S. S. Y. Ho, C. Chua, L. Gole, A. Biswas, E. Koay and
M. Choolani, Prenatal Diagn., 2012, 32, 321–328.
79 E. E. Hayden, Nature, 2012, 486, 454.
80 KellBenx, Inc. www.kellbenx.com. 81 Kellbenx Inc, J. Khosravi, L. H. Kellner and H. Benanni,
Patent US, 2013/0122492 A1.82 Silicon Biosystems S.p.A. www.siliconbiosystems.com.
83 CellScape Corp. www.cellscapecorp.com. 84 CellScape Corp, B. Parikh, M. D. Brody, J. Stone, B. Awabdy,
U. Ved, A. Tran, P. J. Collins and D. J. Anvar, Patent WO,2013075100 A1.

Lab Chip, 2014, 14, 841–854 | 853



Publ

ishe

d on

23

Janu

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View Article Online

85 Verinata Health, Inc. www.verinata.com/.
86 A. J. Sehnert, B. Rhees, D. Comstock, E. de Feo, G. Heilek,
J. Burke and R. P. Rava, Clin. Chem., 2011, 57, 1042–1049.87 D. W. Bianchi, L. D. Platt, J. D. Goldberg, A. Z. Abuhamad,

A. J. Sehnert, R. P. Rava and MELISSA Study Group, Obstet.Gynecol., 2012, 119, 890–901.

88 Sequenom, Inc. www.sequenom.com/.
89 M. Ehrich, C. Deciu, T. Zwiefelhofer, J. A. Tynan,
L. Cagasan, R. Tim, V. Lu, R. McCullough, E. McCarthy,A. O. Nygren, J. Dean, L. Tang, D. Hutchison, T. Lu,H. Wang, V. Angkachatchai, P. Oeth, C. R. Cantor,A. Bombard and D. van den Boom, Am. J. Obstet. Gynecol.,2011, 204(3), 205.e1–11.

90 A. T. Bombard, R. Akolekar, D. H. Farkas, A. L. VanAgtmael,
F. Aquino, P. Oeth and K. H. Nicolaides, Prenatal Diagn.,2011, 31, 802–808.
91 G. E. Palomaki, C. Deciu, E. M. Kloza, G. M. Lambert-
Messerlian, J. E. Haddow, M. Neveux, M. Ehrich, D. vanden Boom, A. T. Bombard, W. W. Grody, S. F. Nelson andJ. A. Canick, Genet. Med., 2012, 14, 296–305.
92 Ariosa Diagnostics, Inc. www.ariosadx.com/.
93 G. Ashoor, A. Syngelki, M. Wagner, C. Birdir and
K. H. Nicolaides, Am. J. Obstet. Gynecol., 2012, 206(4), 322.e1–322.e5.

94 M. E. Norton, H. Brar, J. Weiss, A. Karimi, L. C. Laurent,
A. B. Caughey, M. H. Rodriguez, J. Williams, M. E. Mitchell,C. D. Adair, H. Lee, B. Jacobsson, M. W. Tomlinson,D. Oepkes, D. Hollemon, A. A. Sparks, A. Oliphant andK. Song, Am. J. Obstet. Gynecol., 2012, 207(2), 137.e1–8.
95 K. H. Nicolaides, A. Syngelaki, G. Ashoor, C. Birdir and
G. Touzet, Am. J. Obstet. Gynecol., 2012, 207(5), 374.e1–6.
96 Natera, Inc. http://www.natera.com/.
97 B. Zimmermann, M. Hill, G. Gemelos, Z. Demko, M. Banjevic,
J. Baner, S. Sigurjonsson, N. Chopra, M. Dodd, B. Levy andM. Rabinowitz, Prenatal Diagn., 2012, 32, 1233–1242.

98 Berry Genomics Co. www.berrygenomics.com.

854 | Lab Chip, 2014, 14, 841–854

99 Abnova Co. www.abnova.com.
100 Ravgen Inc. www.ravgen.com. 101 NIPD genetics Ltd. www.nipd.com. 102 BGI Health www.genomics.cn. 103 M. T. Mennuti, A. M. Cherry, J. J. D. Morrissette and
L. Dugoff, Am. J. Obstet. Gynecol., 2013, 209, 415–419.104 R. E. Reiss and A. M. Cherry, Am. J. Obstet. Gynecol., 2013,

209, 160–161.105 The American College of Obstetricians and Gyanecologists

Committee on Genetics, Committee Opinion, No 545, Obstet.Gynecol., 2012, 120, 1532–4.

106 J. A. Canick, G. E. Palomki, E. M. Kloza, G. M. Lambert-
Messerlian and J. E. Haddow, Prenatal Diagn., 2013, 33,667–674.
107 E. M. J. Boon and B. H. W. Faas, Prenatal Diagn., 2013, 33,
563–568.
108 A. Agarwal, L. C. Sayres, M. K. Cho, R. Cook-Deegan and
S. Chandrasekharan, Prenatal Diagn., 2013, 33, 521–531.
109 A. A. S. Bhagat and C. T. Lim, Recent Results Cancer Res.,
2012, 59–67.
110 S. H. Cho, C. H. Chen, F. S. Tsai, J. M. Godin and Y. H. Lo,
Miniaturisation for Chemistry and Biology, Lab Chip, 2010,10, 1567–1573.
111 D. Bianchi and J. Hanson, J. Matern.–Fetal Neonat. Med.,
2006, 19, 199–207.
112 H. W. Wu, C. C. Lin and G. B. Lee, Biomicrofluidics, 2012, 5.
113 G. S. Dawe, X. W. Tan and Z. C. Xiao, Cell Adhesion and
Migration, 2007, 1, 19–27.114 N. Oomman and B. R. Ganatra, Reproductive Health

Matters, 2002, 10, 184–188.115 P. A. Benn and A. R. Chapman, Curr. Opin. Obstet. Gynecol.,

2010, 22, 128–134.116 D. Schmitz, C. Netzer and W. Henn, Nat. Rev. Genet., 2009,

10, 515.117 V. Ravitsky, Nat. Rev. Genet., 2009, 10, 733.
118 C. B. Mitchell, Genetic Ethics, 1997.


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Lab-on-a-chip technology: impacting non-invasive prenatal diagnostics (NIPD) through miniaturisation