it has been roughly half a century since lejeune et al1 first described down syndrome in 1959 . while the technology employed for prenatal 
 detection of down syndrome has expanded in leaps and bounds , the primary focus of prenatal care 
 remains the same : to offer women the most thorough risk assessment with the least invasive procedure 
 possible . down syndrome is the leading cause of prenatal chromosome abnormalities , accounting for 
 53% of all reported chromosome conditions.2 testing strategies , guidelines , and screening options have expanded from their conception in the 
 1970s . at that time , any woman aged 35 years or older was considered to be of advanced maternal age , 
 and this was the sole criteria used by the american congress of obstetricians and gynecologists to 
 define pregnancies that should be offered amniocentesis or chorionic villus sampling . as of 2007 , 
 the american congress of obstetricians and gynecologists has defined a pregnancy as 
  high - risk  when any of the following criteria are met : family history of 
 aneuploidy , advanced maternal age , abnormal serum screen , or abnormal ultrasound findings.3,4 
 while multiple screening options are currently available , the only diagnostic tests offered 
 prenatally for down syndrome are amniocentesis and chorionic villus sampling . this review compares 
 the risks , benefits , and limitations of all currently available prenatal screening methods for 
 detection of down syndrome . anatomical ultrasound has been used since the 1980s to provide health care practitioners and 
 expectant mothers with information regarding a pregnancy.5 with advances in technology , prenatal sonography has expanded from focusing on detection 
 of major structural abnormalities ( ie , cardiac defects , hydrops , duodenal atresia , or cystic 
 hygroma ) to include detection of soft markers to assist in identifying pregnancies that are at risk 
 of various chromosomal conditions . bricker et al6 have defined soft markers as 
  structural changes detected at ultrasound scan which may be transient and in themselves 
 have little or no pathological significance , but are thought to be more commonly found in fetuses 
 with congenital abnormalities , particularly karyotypic abnormalities.46 soft markers 
 that have been linked to down syndrome include nuchal thickening , echogenic intracardiac focus , 
 echogenic bowel , renal pelvic dilation , shortened long bones , absence of the nasal bone , 
 pyelectasis , ventriculomegaly , clinodactyly , and sandal gap toe.5,7,8 in the absence of soft markers , the sensitivity of 
 anatomical ultrasound to detect down syndrome is relatively low at 50% . however , the 
 presence of one soft marker is associated with an increased risk of down syndrome , ie , one soft 
 marker increases the risk by two - fold and three or more soft markers increases the risk by 
 100-fold.7 many scoring indices have been created 
 to help maximize sensitivity , while decreasing false - positive rates.7,9,10 these indices incorporate the presence of structural 
 abnormalities and/or soft markers and maternal age to provide physicians with a guideline as to who 
 should be offered more invasive procedures . while these guidelines have assisted in determining the 
 criteria for a positive finding , they remain limited by the quality of the ultrasound and the 
 expertise of the sonographer . beginning in 1984 , multiple marker screening provided physicians with a means of offering an 
 individualized risk for down syndrome without the inherent risk imposed by chorionic villus sampling 
 or amniocentesis ( table 1).11,12 this second 
 trimester screening , performed at 1520 weeks gestation , is often referred to as the quad 
 screen because it incorporates maternal age - related risk and four maternal serum biomarkers , ie , 
 alpha - fetoprotein , free beta human chorionic gonadotropin , unconjugated estriol , and dimeric inhibin 
 a levels.11 by combining maternal age with the 
 quad screen , the detection rate is roughly 75% for down syndrome in women younger than 35 
 years and > 80% in women 35 years and older ( with a positive screening rate of 
 5%).12 it was not until the late 1990s that first trimester screening was introduced as an earlier 
 screening option for the detection of down syndrome . first trimester screening incorporates maternal 
 age , nuchal translucency ultrasonography , and measurement of maternal serum free beta human 
 chorionic gonadotropin and pregnancy - associated plasma protein a.3,4,12,13 
 collection of blood for biochemical analysis and ultrasound assessment for nuchal translucency is 
 typically performed between 11 and 13 6/7 weeks gestation . increased nuchal translucency , reduction 
 in pregnancy - associated plasma protein a levels , and an increase in beta human chorionic 
 gonadotropin can be an indication of down syndrome , and assist practitioners in identifying 
 pregnancies at risk for the syndrome . a nuchal translucency measurement by itself has a detection 
 rate for down syndrome of about 70% with a 5% false - positive rate , but when combined 
 with pregnancy - associated plasma protein a and beta human chorionic gonadotropin measurements , 
 detection rates increase to 79%90% , with a 5% false - positive screen 
 rate.11,14 various studies have been conducted to determine the optimal time for performing the 
 first trimester screening , with the goal of providing a maximum detection rate while still 
 maintaining a low false - positive rate.1518 these studies suggest that 
 earlier pregnancy - associated plasma protein a and beta human chorionic gonadotropin measurements 
 taken at 910 weeks gestation , with nuchal translucency measurement taken at 12 weeks 
 gestation , can increase the detection rate to 90%93% , with a 
 3%5% false - positive rate.15,17,18 a detection rate of 92%95% with a 
 3%5% false - positive rate can be achieved when pregnancy - associated plasma 
 protein a measurements are done at 910 weeks gestation , with beta human chorionic 
 gonadotropin and nuchal translucency measurements taken at 12 weeks gestation.15 first trimester screening gives women who receive prenatal care prior 
 to 14 weeks gestation the ability to have information sooner than with second trimester screening . if the results reveal an increased risk of fetal aneuploidy , the woman can be offered genetic 
 counseling with the option to choose either first - trimester chorionic villus sampling or 
 second - trimester amniocentesis . independent sequential screening is defined as independently performed first - trimester and 
 second - trimester screenings , with separate individualized risk assessments given.11,19 
 although independent sequential screening increases the detection rate from 
 88%91% to 94% , it also increases the false - positive rate from 
 5% to 11%.11,19 thus , it is recommended that women who undergo first - trimester 
 screening for aneuploidy should not also have second - trimester serum screening in the same 
 pregnancy . if a higher detection rate is preferred , an integrated or sequential screening test which 
 combines both first - trimester and second - trimester screening is suggested . integrated screening is defined as the process by which a patient s individualized risk 
 is calculated based on the combination of both the first - trimester and second - trimester 
 screenings.11,19 due to the nature of this screening method , a patient s 
 first - trimester screening results are not disclosed until second - trimester screening is performed 
 and a combined risk based on both screenings can be calculated.11,19 there has been 
 ethical debate regarding integrated screening because the patient s first - trimester 
 screening results are not disclosed until the conclusion of the second - trimester screening . this 
 precludes patients who are at high risk based on first - trimester screening from being offered 
 chorionic villus sampling and having more options available to them . in contrast , patients undergoing stepwise sequential screening are provided with their individual 
 risk once the results from the first - trimester screen are available.11,19 patients who 
 screen positive during first - trimester screening are offered genetic counseling and are given the 
 information regarding chorionic villus sampling and amniocentesis . patients who screen negative are 
 offered second - trimester screening and are provided with an adjusted risk number that incorporates 
 both the first - trimester and second - trimester results . incorporation of both the first - trimester and 
 second - trimester screenings in this stepwise manner increases the detection rate to 
 94%96% while still maintaining a low false - positive rate of 
 5%6%.11,19 the final type of sequential screening is known as the contingent screening method . this method 
 is similar to stepwise sequential screening in that scores are calculated based on results from both 
 the first - trimester and second - trimester screening.11,19 however , the contingent screening 
 method uses the first - trimester results to classify patients into three subgroups , ie , 
 screen - positive , screen - negative , and borderline.11,19 second - trimester screening is only 
 offered to patients who fall into either the screen - negative or the borderline group . the detection 
 rate for this method is 94%95% , with a false - positive rate of about 
 5%.11,19 it has been known since the mid 1950s that fetal cells are present in the maternal 
 circulation.20,21 however , low yield ( 1 fetal cell / ml of maternal blood ) , inability to 
 develop an efficient enrichment process , and the tendency for fetal dna to disintegrate during 
 chromosome extraction have inhibited utilization of these fetal cells for the development of a 
 noninvasive prenatal test.20,2224 use of 
 noninvasive prenatal testing targeted to fetal cells was finally abandoned when bianchi et al25 demonstrated that fetal cells could remain in the 
 maternal circulation for decades , thus making it impossible to distinguish new fetal cells from 
 those of previous pregnancies . it was not until 1997 when lo et al26 
 demonstrated the existence of cell - free fetal dna in the maternal circulation that the scientific 
 community was presented with a new possible target for noninvasive prenatal testing . fetal cells , 
 theorized to be derived from the placenta , enter the maternal circulation where they undergo 
 apoptosis . in the process , fetal dna is cleaved into small 150200 base pairs fragments , 
 which are then released into the maternal bloodstream.20 these cell - free fetal dna fragments can be detected as early as the fourth week of 
 gestation and reliably after the seventh week of gestation.12,20,23 unlike the longevity seen with fetal cells , cell - free 
 fetal dna has a half - life of only 16 minutes and is cleared from the maternal circulation within 2 
 hours of delivery.27 the low false - positive rate 
 as well as the prevalence of cell - free fetal dna in the maternal circulation ( accounting for 
 > 10% of all cell - free dna or 1680 fetal genomes / ml of maternal blood ) , made 
 cell - free fetal dna a desirable target for noninvasive prenatal testing.12,20,23,28,29 over the next 15 years , scientists would work on developing and refining methods of detecting 
 pregnancies at risk for aneuploidy . in october , 2011 , sequenom inc ( san diego , ca , usa ) was the 
 first company to make a noninvasive pre - natal test commercially available for the detection of down 
 syndrome . to date , three companies ( verinata health [ redwood , ca , usa ] , ariosa 
 diagnostics [ san jose , ca , usa ] , and sequenom inc ) offer a noninvasive prenatal test 
 for the detection of trisomies 13 and 18 and down syndrome , with a fourth company ( natera , san 
 carlo , ca , usa ) expected to have a commercially available noninvasive prenatal test within the next 
 few months ( figure 1 ) . figure 1 describes the various strategies 
 employed by the four companies to obtain and analyze maternal and/or fetal dna from the maternal 
 serum sample . two companies , sequenom inc and verinata health , utilize massively parallel shotgun 
 sequencing , commonly referred to as shotgun sequencing,3031 while the other two 
 companies , natera and ariosa diagnostics , utilize targeted sequence analysis.3336 the main 
 difference between these two technologies is that shotgun sequencing results in amplification of all 
 genetic information , while targeted sequencing results in amplification of only the genetic 
 information of interest , ie , chromosome(s ) . targeted sequencing enables companies such as natera and 
 ariosa diagnostics to perform noninvasive prenatal testing on smaller sample sizes compared with 
 sequenom inc and verinata health . massively parallel shotgun sequencing technology requires more 
  reads  ( or dna fragments ) than those required by targeted sequencing to ensure that 
 there are enough fragments from the chromosomes of interest to provide accurate results . a major 
 disadvantage of targeted sequencing is the inability to rule out other chromosomal conditions , such 
 as microduplications and deletions , because they have not been selected for . while this does not 
 seem to be a major limiting factor at this time , due to the fact that companies are not currently 
 reporting nonvalidated findings , it may become a shortcoming in the future as companies aim to 
 expand noninvasive prenatal testing to include other chromosomal abnormalities , such as 
 microdeletions and microduplications . all four companies currently use variations of two methodologies for analyzing genetic data , ie , 
 quantitative read counting or single nucleotide polymorphism detection . three of the companies 
 ( sequenom inc , verinata health , and ariosa diagnostics ) use quantitative read counting , while natera 
 uses bioinformatic algorithms to analyze single nucleotide polymorphism data obtained by 
 next - generation aneuploidy testing using single nucleotide polymorphisms . quantitative read counting analyzes the number of chromosome fragments present from each 
 chromosome or chromosomes of interest . because the amount of genetic material from each chromosome 
 is directly proportional to the chromosome size , companies can use a known euploid reference sample 
 to calculate the expected proportion of genetic information from each chromosome given a euploid 
 pregnancy.31,32,3739 for example , chromosome 21 generally accounts for 
 1.5% of the human genome.31,3739 any change in the actual proportions of genetic information from chromosome 21 that is 
 2.53.1 standard deviations from the mean is determined to be an aneuploidy.30,31,40 the benefit of this technology is 
 that it does not require any differentiation between maternal and fetal genetic information . however , this is also a limitation resulting from the fact that , in a fetus with down syndrome , the 
 contribution of genetic information from chromosome 21 increases from 1.5% to 2.25% , 
 for an overall change of 0.75% . when taking into account the fact that fetal dna only 
 represents 10% of the total dna in the sample , the overall change in the amount of 
 chromosome 21 cell - free - dna would only increase from 1.5% to 1.575% . when the fetal 
 fraction is below 10% , it can result in inconclusive results , or a no - call . one company 
 using quantitative read counting , ariosa diagnostics , has aimed to address this issue by creating 
 the forte ( fraction optimized risk of trisomy evaluation ) assay.33,34 this assay 
 utilizes polymorphic and nonpolymorphic regions that are known to differ between fetal and maternal 
 dna to determine the fetal fraction as well as the overall proportional representation of 
 chromosomal fragments . unlike quantitative read counting , the next - generation aneuploidy testing using single nucleotide 
 polymorphisms system created by natera utilizes the parental support statistical algorithm 
 to analyze the sequenced fetal cell - free dna together with maternal genetic information , paternal 
 genetic information , and hapmap technology.35,36 this algorithm provides a series of 
 possible  hypotheses  of fetal genotypes ( eg , monosomy , disomy , trisomy ) based on 
 known common crossover points on the chromosome(s ) of interest , parental information , fetal 
 fraction , and fetal chromosome copy number . the sample - specific confidence interval for each 
 hypothesis is calculated , and a  hypothesis  is considered confirmed when the 
 confidence interval is above 98%.36 while the results seen from these four companies are promising ( table 2 ) , it is important to remember that noninvasive prenatal testing 
 is for screening and is not a diagnostic tool.30,32,35,36,41 noninvasive prenatal test results group patients into 
 three possible categories , ie , low risk of aneuploidy , high risk of aneuploidy , or no - call 
 ( undeterminable ) . the clinical implications for low - risk and high - risk populations are the same for 
 noninvasive prenatal testing as they are for first - trimester screening and multiple marker 
 screening . individuals who are determined to be in the low - risk category would not require any 
 further diagnostic evaluation , while those in the high - risk category are recommended to undergo 
 diagnostic testing ( ie , chorionic villus sampling or amniocentesis ) . individuals who fall into the 
 third category of  no - call  would require a repeat sample to be drawn . for these 
 reasons , noninvasive prenatal testing is not intended to serve as a replacement for amniocentesis or 
 chorionic villus sampling , but rather a methodology that would enable fewer unnecessary diagnostic 
 procedures by detecting patients who are at high risk for aneuploidy more accurately , when compared 
 with the detection rates for first - trimester screening and multiple marker screening . physicians 
 should ensure that their patients understand that these tests are not designed to circumvent an 
 unwanted diagnostic procedure , and in actuality may result in the recommendation of such a 
 diagnostic procedure should test results reveal a high risk for aneuploidy . while noninvasive prenatal tests do have an increased specificity and sensitivity compared with 
 first - trimester screening or multiple marker screening , limitations do exist . possible causes for false - positive results could be placental mosaicism , vanishing 
 twin syndrome , or an unidentified maternal condition , such as mosaicism or cancer . secondly , the 
 majority of noninvasive prenatal tests are currently only offered to women with singleton 
 pregnancies who are at high risk for down syndrome because of family history of aneuploidy , advanced 
 maternal age , an abnormal serum screen , or abnormal ultrasound findings . with the exception of 
 ariosa diagnostics , which has conducted validation studies for harmony in the 
 low - risk population , it is important to note that these tests have not been validated in low - risk or 
 multiple gestation populations , and their accuracy is unknown at this time.42 anatomical ultrasound has been used since the 1980s to provide health care practitioners and 
 expectant mothers with information regarding a pregnancy.5 with advances in technology , prenatal sonography has expanded from focusing on detection 
 of major structural abnormalities ( ie , cardiac defects , hydrops , duodenal atresia , or cystic 
 hygroma ) to include detection of soft markers to assist in identifying pregnancies that are at risk 
 of various chromosomal conditions . bricker et al6 have defined soft markers as 
  structural changes detected at ultrasound scan which may be transient and in themselves 
 have little or no pathological significance , but are thought to be more commonly found in fetuses 
 with congenital abnormalities , particularly karyotypic abnormalities.46 soft markers 
 that have been linked to down syndrome include nuchal thickening , echogenic intracardiac focus , 
 echogenic bowel , renal pelvic dilation , shortened long bones , absence of the nasal bone , 
 pyelectasis , ventriculomegaly , clinodactyly , and sandal gap toe.5,7,8 in the absence of soft markers , the sensitivity of 
 anatomical ultrasound to detect down syndrome is relatively low at 50% . however , the 
 presence of one soft marker is associated with an increased risk of down syndrome , ie , one soft 
 marker increases the risk by two - fold and three or more soft markers increases the risk by 
 100-fold.7 many scoring indices have been created 
 to help maximize sensitivity , while decreasing false - positive rates.7,9,10 these indices incorporate the presence of structural 
 abnormalities and/or soft markers and maternal age to provide physicians with a guideline as to who 
 should be offered more invasive procedures . while these guidelines have assisted in determining the 
 criteria for a positive finding , they remain limited by the quality of the ultrasound and the 
 expertise of the sonographer . beginning in 1984 , multiple marker screening provided physicians with a means of offering an 
 individualized risk for down syndrome without the inherent risk imposed by chorionic villus sampling 
 or amniocentesis ( table 1).11,12 this second 
 trimester screening , performed at 1520 weeks gestation , is often referred to as the quad 
 screen because it incorporates maternal age - related risk and four maternal serum biomarkers , ie , 
 alpha - fetoprotein , free beta human chorionic gonadotropin , unconjugated estriol , and dimeric inhibin 
 a levels.11 by combining maternal age with the 
 quad screen , the detection rate is roughly 75% for down syndrome in women younger than 35 
 years and > 80% in women 35 years and older ( with a positive screening rate of 
 5%).12 it was not until the late 1990s that first trimester screening was introduced as an earlier 
 screening option for the detection of down syndrome . first trimester screening incorporates maternal 
 age , nuchal translucency ultrasonography , and measurement of maternal serum free beta human 
 chorionic gonadotropin and pregnancy - associated plasma protein a.3,4,12,13 
 collection of blood for biochemical analysis and ultrasound assessment for nuchal translucency is 
 typically performed between 11 and 13 6/7 weeks gestation . increased nuchal translucency , reduction 
 in pregnancy - associated plasma protein a levels , and an increase in beta human chorionic 
 gonadotropin can be an indication of down syndrome , and assist practitioners in identifying 
 pregnancies at risk for the syndrome . a nuchal translucency measurement by itself has a detection 
 rate for down syndrome of about 70% with a 5% false - positive rate , but when combined 
 with pregnancy - associated plasma protein a and beta human chorionic gonadotropin measurements , 
 detection rates increase to 79%90% , with a 5% false - positive screen 
 rate.11,14 various studies have been conducted to determine the optimal time for performing the 
 first trimester screening , with the goal of providing a maximum detection rate while still 
 maintaining a low false - positive rate.1518 these studies suggest that 
 earlier pregnancy - associated plasma protein a and beta human chorionic gonadotropin measurements 
 taken at 910 weeks gestation , with nuchal translucency measurement taken at 12 weeks 
 gestation , can increase the detection rate to 90%93% , with a 
 3%5% false - positive rate.15,17,18 a detection rate of 92%95% with a 
 3%5% false - positive rate can be achieved when pregnancy - associated plasma 
 protein a measurements are done at 910 weeks gestation , with beta human chorionic 
 gonadotropin and nuchal translucency measurements taken at 12 weeks gestation.15 first trimester screening gives women who receive prenatal care prior 
 to 14 weeks gestation the ability to have information sooner than with second trimester screening . if the results reveal an increased risk of fetal aneuploidy , the woman can be offered genetic 
 counseling with the option to choose either first - trimester chorionic villus sampling or 
 second - trimester amniocentesis . independent sequential screening is defined as independently performed first - trimester and 
 second - trimester screenings , with separate individualized risk assessments given.11,19 
 although independent sequential screening increases the detection rate from 
 88%91% to 94% , it also increases the false - positive rate from 
 5% to 11%.11,19 thus , it is recommended that women who undergo first - trimester 
 screening for aneuploidy should not also have second - trimester serum screening in the same 
 pregnancy . if a higher detection rate is preferred , an integrated or sequential screening test which 
 combines both first - trimester and second - trimester screening is suggested . integrated screening is defined as the process by which a patient s individualized risk 
 is calculated based on the combination of both the first - trimester and second - trimester 
 screenings.11,19 due to the nature of this screening method , a patient s 
 first - trimester screening results are not disclosed until second - trimester screening is performed 
 and a combined risk based on both screenings can be calculated.11,19 there has been 
 ethical debate regarding integrated screening because the patient s first - trimester 
 screening results are not disclosed until the conclusion of the second - trimester screening . this 
 precludes patients who are at high risk based on first - trimester screening from being offered 
 chorionic villus sampling and having more options available to them . in contrast , patients undergoing stepwise sequential screening are provided with their individual 
 risk once the results from the first - trimester screen are available.11,19 patients who 
 screen positive during first - trimester screening are offered genetic counseling and are given the 
 information regarding chorionic villus sampling and amniocentesis . patients who screen negative are 
 offered second - trimester screening and are provided with an adjusted risk number that incorporates 
 both the first - trimester and second - trimester results . incorporation of both the first - trimester and 
 second - trimester screenings in this stepwise manner increases the detection rate to 
 94%96% while still maintaining a low false - positive rate of 
 5%6%.11,19 the final type of sequential screening is known as the contingent screening method . this method 
 is similar to stepwise sequential screening in that scores are calculated based on results from both 
 the first - trimester and second - trimester screening.11,19 however , the contingent screening 
 method uses the first - trimester results to classify patients into three subgroups , ie , 
 screen - positive , screen - negative , and borderline.11,19 second - trimester screening is only 
 offered to patients who fall into either the screen - negative or the borderline group . the detection 
 rate for this method is 94%95% , with a false - positive rate of about 
 5%.11,19 it has been known since the mid 1950s that fetal cells are present in the maternal 
 circulation.20,21 however , low yield ( 1 fetal cell / ml of maternal blood ) , inability to 
 develop an efficient enrichment process , and the tendency for fetal dna to disintegrate during 
 chromosome extraction have inhibited utilization of these fetal cells for the development of a 
 noninvasive prenatal test.20,2224 use of 
 noninvasive prenatal testing targeted to fetal cells was finally abandoned when bianchi et al25 demonstrated that fetal cells could remain in the 
 maternal circulation for decades , thus making it impossible to distinguish new fetal cells from 
 those of previous pregnancies . it was not until 1997 when lo et al26 
 demonstrated the existence of cell - free fetal dna in the maternal circulation that the scientific 
 community was presented with a new possible target for noninvasive prenatal testing . fetal cells , 
 theorized to be derived from the placenta , enter the maternal circulation where they undergo 
 apoptosis . in the process , fetal dna is cleaved into small 150200 base pairs fragments , 
 which are then released into the maternal bloodstream.20 these cell - free fetal dna fragments can be detected as early as the fourth week of 
 gestation and reliably after the seventh week of gestation.12,20,23 unlike the longevity seen with fetal cells , cell - free 
 fetal dna has a half - life of only 16 minutes and is cleared from the maternal circulation within 2 
 hours of delivery.27 the low false - positive rate 
 as well as the prevalence of cell - free fetal dna in the maternal circulation ( accounting for 
 > 10% of all cell - free dna or 1680 fetal genomes / ml of maternal blood ) , made 
 cell - free fetal dna a desirable target for noninvasive prenatal testing.12,20,23,28,29 over the next 15 years , scientists would work on developing and refining methods of detecting 
 pregnancies at risk for aneuploidy . in october , 2011 , sequenom inc ( san diego , ca , usa ) was the 
 first company to make a noninvasive pre - natal test commercially available for the detection of down 
 syndrome . to date , three companies ( verinata health [ redwood , ca , usa ] , ariosa 
 diagnostics [ san jose , ca , usa ] , and sequenom inc ) offer a noninvasive prenatal test 
 for the detection of trisomies 13 and 18 and down syndrome , with a fourth company ( natera , san 
 carlo , ca , usa ) expected to have a commercially available noninvasive prenatal test within the next 
 few months ( figure 1 ) . figure 1 describes the various strategies 
 employed by the four companies to obtain and analyze maternal and/or fetal dna from the maternal 
 serum sample . two companies , sequenom inc and verinata health , utilize massively parallel shotgun 
 sequencing , commonly referred to as shotgun sequencing,3031 while the other two 
 companies , natera and ariosa diagnostics , utilize targeted sequence analysis.3336 the main 
 difference between these two technologies is that shotgun sequencing results in amplification of all 
 genetic information , while targeted sequencing results in amplification of only the genetic 
 information of interest , ie , chromosome(s ) . targeted sequencing enables companies such as natera and 
 ariosa diagnostics to perform noninvasive prenatal testing on smaller sample sizes compared with 
 sequenom inc and verinata health . massively parallel shotgun sequencing technology requires more 
  reads  ( or dna fragments ) than those required by targeted sequencing to ensure that 
 there are enough fragments from the chromosomes of interest to provide accurate results . a major 
 disadvantage of targeted sequencing is the inability to rule out other chromosomal conditions , such 
 as microduplications and deletions , because they have not been selected for . while this does not 
 seem to be a major limiting factor at this time , due to the fact that companies are not currently 
 reporting nonvalidated findings , it may become a shortcoming in the future as companies aim to 
 expand noninvasive prenatal testing to include other chromosomal abnormalities , such as 
 microdeletions and microduplications . all four companies currently use variations of two methodologies for analyzing genetic data , ie , 
 quantitative read counting or single nucleotide polymorphism detection . three of the companies 
 ( sequenom inc , verinata health , and ariosa diagnostics ) use quantitative read counting , while natera 
 uses bioinformatic algorithms to analyze single nucleotide polymorphism data obtained by 
 next - generation aneuploidy testing using single nucleotide polymorphisms . quantitative read counting analyzes the number of chromosome fragments present from each 
 chromosome or chromosomes of interest . because the amount of genetic material from each chromosome 
 is directly proportional to the chromosome size , companies can use a known euploid reference sample 
 to calculate the expected proportion of genetic information from each chromosome given a euploid 
 pregnancy.31,32,3739 for example , chromosome 21 generally accounts for 
 1.5% of the human genome.31,3739 any change in the actual proportions of genetic information from chromosome 21 that is 
 2.53.1 standard deviations from the mean is determined to be an aneuploidy.30,31,40 the benefit of this technology is 
 that it does not require any differentiation between maternal and fetal genetic information . however , this is also a limitation resulting from the fact that , in a fetus with down syndrome , the 
 contribution of genetic information from chromosome 21 increases from 1.5% to 2.25% , 
 for an overall change of 0.75% . when taking into account the fact that fetal dna only 
 represents 10% of the total dna in the sample , the overall change in the amount of 
 chromosome 21 cell - free - dna would only increase from 1.5% to 1.575% . when the fetal 
 fraction is below 10% , it can result in inconclusive results , or a no - call . one company 
 using quantitative read counting , ariosa diagnostics , has aimed to address this issue by creating 
 the forte ( fraction optimized risk of trisomy evaluation ) assay.33,34 this assay 
 utilizes polymorphic and nonpolymorphic regions that are known to differ between fetal and maternal 
 dna to determine the fetal fraction as well as the overall proportional representation of 
 chromosomal fragments . unlike quantitative read counting , the next - generation aneuploidy testing using single nucleotide 
 polymorphisms system created by natera utilizes the parental support statistical algorithm 
 to analyze the sequenced fetal cell - free dna together with maternal genetic information , paternal 
 genetic information , and hapmap technology.35,36 this algorithm provides a series of 
 possible  hypotheses  of fetal genotypes ( eg , monosomy , disomy , trisomy ) based on 
 known common crossover points on the chromosome(s ) of interest , parental information , fetal 
 fraction , and fetal chromosome copy number . the sample - specific confidence interval for each 
 hypothesis is calculated , and a  hypothesis  is considered confirmed when the 
 confidence interval is above 98%.36 while the results seen from these four companies are promising ( table 2 ) , it is important to remember that noninvasive prenatal testing 
 is for screening and is not a diagnostic tool.30,32,35,36,41 noninvasive prenatal test results group patients into 
 three possible categories , ie , low risk of aneuploidy , high risk of aneuploidy , or no - call 
 ( undeterminable ) . the clinical implications for low - risk and high - risk populations are the same for 
 noninvasive prenatal testing as they are for first - trimester screening and multiple marker 
 screening . individuals who are determined to be in the low - risk category would not require any 
 further diagnostic evaluation , while those in the high - risk category are recommended to undergo 
 diagnostic testing ( ie , chorionic villus sampling or amniocentesis ) . individuals who fall into the 
 third category of  no - call  would require a repeat sample to be drawn . for these 
 reasons , noninvasive prenatal testing is not intended to serve as a replacement for amniocentesis or 
 chorionic villus sampling , but rather a methodology that would enable fewer unnecessary diagnostic 
 procedures by detecting patients who are at high risk for aneuploidy more accurately , when compared 
 with the detection rates for first - trimester screening and multiple marker screening . physicians 
 should ensure that their patients understand that these tests are not designed to circumvent an 
 unwanted diagnostic procedure , and in actuality may result in the recommendation of such a 
 diagnostic procedure should test results reveal a high risk for aneuploidy . while noninvasive prenatal tests do have an increased specificity and sensitivity compared with 
 first - trimester screening or multiple marker screening possible causes for false - positive results could be placental mosaicism , vanishing 
 twin syndrome , or an unidentified maternal condition , such as mosaicism or cancer . secondly , the 
 majority of noninvasive prenatal tests are currently only offered to women with singleton 
 pregnancies who are at high risk for down syndrome because of family history of aneuploidy , advanced 
 maternal age , an abnormal serum screen , or abnormal ultrasound findings . with the exception of 
 ariosa diagnostics , which has conducted validation studies for harmony in the 
 low - risk population , it is important to note that these tests have not been validated in low - risk or 
 multiple gestation populations , and their accuracy is unknown at this time.42 it has long been a goal of prenatal genetic diagnosis to develop a noninvasive test that would 
 allow for detection of aneuploidy and eliminate the need for invasive testing , such as chorionic 
 villus sampling or amniocentesis . while this is still not currently possible , developments in 
 noninvasive prenatal testing are the initial steps in this direction . while the scope of this review 
 does not include other aneuploidy conditions , all of the companies discussed do offer screening for 
 trisomy 13 and 18 , with varying degrees of success . the noninvasive prenatal tests marketed by 
 verinata health and natera also include a screen for sex chromosome abnormalities , such as turner 
 syndrome , kleinfelter syndrome , and 47 , xyy syndrome . validation studies are currently underway for 
 noninvasive prenatal testing in the detection of fetal aneuploidies in the general population as 
 well as multiple gestations . furthermore , the future of noninvasive prenatal testing may expand 
 beyond the common aneuploidies to include other chromosomal conditions , such as microdeletions and 
 microduplications . with the ever - expanding testing options that are now available to the expectant 
 mother , it is imperative that physicians remain up to date on these technologies and have a clear 
 understanding of the risks , benefits , and limitations of these technologies . by fully understanding 
 each technology and the possible alternatives , physicians can provide their patients with all the 
 information necessary to make an informed decision regarding medical management .
down syndrome is the leading cause of prenatal chromosome abnormalities , accounting for 
 53% of all reported chromosome conditions . testing strategies , guidelines , and screening 
 options have expanded from their conception in the 1970s , and now include such options as anatomical 
 ultrasound , maternal serum screening , and noninvasive prenatal testing . 
 this review summarizes all 
 currently available noninvasive diagnostic techniques for the detection of down syndrome . by 
 understanding fully each technology and the possible alternatives 
 , the physician will be able to 
 provide their patients with all the information necessary to make an informed decision regarding 
 their medical management .