http://informahealthcare.com/jmfISSN: 1476-7058 (print), 1476-4954 (electronic)

J Matern Fetal Neonatal Med, Early Online: 1–6! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.947579

ORIGINAL ARTICLE

Initial experience with non-invasive prenatal testing of cell-free DNA formajor chromosomal anomalies in a clinical setting

Carmina Comas, Monica Echevarria, M Angeles Rodrıguez, Pilar Prats, Ignacio Rodrıguez, and Bernat Serra

Fetal Medicine Unit, Department of Obstetrics and Gynecology, Hospital Universitari Quiron Dexeus, Barcelona, Spain

Abstract

Objective: To evaluate non-invasive prenatal testing (NIPT) of cell-free DNA (cfDNA) as ascreening method for major chromosomal anomalies (CA) in a clinical setting.Methods: From January to December 2013, Panorama� test or Harmony� prenatal test wereoffered as advanced NIPT, in addition to first-trimester combined screening in singletonpregnancies.Results: The cohort included 333 pregnant women with a mean maternal age (MA) of 37 yearswho underwent testing at a mean gestational age of 14.6 weeks. Eighty-four percent were low-risk pregnancies. Results were provided in 97.3% of patients at a mean reporting time of 12.9calendar days. Repeat sampling was performed in six cases and results were obtained in five ofthem. No results were provided in four cases. Four cases of Down syndrome were detected andthere was one discordant result of Turner syndrome. We found no statistical differencesbetween commercial tests except in reporting time, fetal fraction and MA. The cfDNA fractionwas statistically associated with test type, maternal weight, BMI and log bhCG levels.Conclusions: NIPT has the potential to be a highly effective screening method for major CA in aclinical setting.

Keywords

Cell-free DNA, chromosomal anomalies,first-trimester screening, non-invasiveprenatal testing, prenatal diagnosis,trisomy 21

History

Received 11 June 2014Accepted 20 July 2014Published online 12 August 2014

Introduction

The recent development of non-invasive prenatal testing

(NIPT) for aneuploidies marks the beginning of a new era for

prenatal screening. Several recent studies have demonstrated

that the most effective method of NIPT for trisomy 21, with

detection rates499% and false-positive rates (FPR) of �0.1%,

is derived from the examination of cell-free DNA (cfDNA) in

maternal plasma [1–8]. Conventional first-trimester combined

screening (FTS) including serum markers and nuchal trans-

lucency has 85–90% detection rates with 5% FPRs [9–11].

Consequently, cfDNA testing is far superior to screening

methods that are currently in use, and should lead to

widespread future use of the test in routine clinical practice.

There are two different approaches to analyzing cfDNA to

screen for chromosomal anomalies (CA): quantitative and

single-nucleotide polymorphism (SNP)-based methods. In the

first approach, maternal plasma cfDNA molecules are

sequenced and the chromosomal origin of each molecule is

identified by comparing it with the human genome. In

trisomic pregnancies, the quantity of molecules derived from

the trisomic chromosome, as compared with an assumed

disomic reference chromosome, is higher than in euploid

pregnancies. In addition to random massively parallel shot-

gun sequencing (MPSS) technology [1–3,7,12–17], targeted

massively parallel sequencing (t-MPS) has been successfully

applied, reducing costs and increasing efficiency [4–6,8,

18–20]. In contrast, SNP-based methods determine the

number of chromosomal copies by looking for specific

patterns in allelic measurements [21,22]. Previously published

experiences have demonstrated advantages and limitations to

both approaches, but there is still a lack of evidence to

definitively support either one of them. Consequently, current

scientific opinion is not clearly positioned with respect to a

preferred technique.

Regardless of the approach, the ability to complete the

analysis and thus obtain a reliable clinical result is related to

the proportion of fetal to maternal cfDNA in maternal plasma,

where the minimum cfDNA needed for analysis is �4% [6].

Lower fetal fractions correlate with an increased error rate,

particularly in counting methods. Other than gestational age

(GA) and maternal weight [23,24], little is known about the

clinical and biologic factors influencing this parameter.

We describe our initial 12-month experience prospectively

implementing NIPT in a private practice. Our primary aim

was to report our experience in the introduction of this new

technology in a real clinical setting. A secondary aim was to

compare cfDNA NIPT for aneuploidies using different tests.

And finally, we also tried to assess clinically significant

factors influencing cfDNA fetal fraction.

Address for correspondence: Carmen Comas Gabriel, Fetal MedicineUnit, Department of Obstetrics and Gynecology, Hospital UniversitariQuiron Dexeus, Gran Vıa Carles III 71-75, Barcelona 08028, Spain. Tel:0034-932274706. Fax: 934187832. E-mail: carcom@dexeus.com

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Methods

Study population

This was an observational prospective study of singleton

pregnancies that underwent prenatal screening for fetal

trisomy from January to December 2013 in a private prenatal

diagnostics center in Barcelona, Spain. As it is the standard of

care in our country, FTS consisting of pregnancy-associated

plasma protein-A (PAPP-A) serum level and free b-human

chorionic gonadotropin (b-hCG) combined with nuchal

translucency measurement was offered to all pregnant

women. Since January 2013, our center has offered NIPT

with cfDNA in addition to FTS for all singleton pregnancies,

excluding cases of ultrasound anomalies (fetal structural

anomalies or NT above the 99th percentile) or those at high

risk of other genetic conditions.

The Panorama� test (Natera, Inc., San Carlos, CA) and

the Harmony� prenatal test (Ariosa Diagnostics, Inc., San

Jose, CA) were offered as advanced NIPT options. During the

first 4 months, only the Harmony� prenatal test for trisomy

13, 18 and 21 was ready for use. Since then, both tests and

testing for sex chromosome abnormalities have become

available. If patients chose NIPT in addition to the FTS,

testing was performed from Week 9 for the Panorama test or

from Week 10 for the Harmony test. All women received

genetic counseling from a fetal medicine specialist and

provided written informed consent prior to testing. High-risk

results from NIPT were confirmed by karyotyping through

invasive testing. Complete follow-up was considered when

invasive testing was performed or neonatal data was obtained.

Pretest counseling

During pretest counseling, women were told that the cfDNA

test was a high-performance screening test rather than a

diagnostic test. It was noted that in �3–5% of cases the test

might be inconclusive; in those cases, trisomy risk would be

determined from the results of the FTS. The parents were

informed that if the 11–14-week ultrasound showed a high NT

thickness (greater than the 99th percentile) or any major

defects it might still be advisable to consider invasive testing.

Laboratory analysis

Up to 20 ml of whole blood were collected via standard

venipuncture into two cfDNA BCTTM tubes (Streck, Omaha,

NE), after completion of written informed consent. Samples

were sent to the laboratories via same day courier (Natera,

Inc. or LABCO, Ariosa Diagnostics, Inc.), at ambient

temperature without any further processing. If the

Panorama� test was chosen, paternal buccal samples were

collected when possible. For each case, the following

information was provided to the laboratory: maternal age

(MA), GA, reason for referral and date of collection.

Statistical analysis

Descriptive data were presented as mean values with range

and percentages for categorical variables. The Wilcoxon

signed-rank test was used to compare means between groups

and the �2 or Fisher’s exact tests were used for categorical

variables. The correlation between fetal fraction and

continuous variables was evaluated using the Spearman’s

correlation coefficient. All tests were bilateral with a 0.05

significance level.

Results

Patient characteristics

The population included 333 pregnant women with a mean

MA of 37 years (range 21–46 years) who underwent testing at

a mean GA of 14.6 weeks (range 9.5–23.5 weeks) (Table 1).

Sex chromosome abnormalities were tested for in 217 cases

(65%). Regarding indication for NIPT, 83.5% were low-risk

pregnancies referred by anxiety and 16.5% were high-risk

pregnancies (11.4% of them referred for high risk at FTS).

Paternal buccal samples were collected in 109 (51%) of the

Panorama cases. Complete follow-up was done in 315 cases

(94.5%). The remaining pregnancies are currently in progress.

Test performance

There was no significant technical or logistical problem

encountered with the implementation of these tests. The NIPT

results were provided for 97.3% of patients at a mean

reporting time of 12.9 (8–21) calendar days. Repeated

sampling was performed in six cases (1.8%) and results

were obtained in five (83%) of them. No results were provided

in four cases (1.2%). Excluding CA, the success rate was

dependent on cfDNA fraction (mean of 12.8% in successful

results versus 7.3% in unsuccessful results) and MA (37.1

versus 39.5 years, respectively). We could not demonstrate

any relationship with body mass index (BMI), PAPP-A levels,

GA or b-hCG levels. With NIPT, four (1.2%) patients were at

high risk of Down syndrome (DS) and one (0.5%) patient was

at high risk of Turner syndrome. In DS cases, all except one

had received positive result by prior FTS. In the remaining

case, NIPT was performed as a primary screening test for

advanced MA (although she had also tested positive after the

NIPT). All four DS cases were correctly identified (100%

detection rate) and there was one discordant result for Turner

syndrome (0.3% FPR). For the 94.5% of cases that had

already been delivered, no false negatives were detected. Fetal

sex was correctly identified in all cases. Description of ‘‘high

risk’’ and ‘‘no result’’ cases with NIPT is shown in Table 2.

Differences between Harmony� and Panorama� tests

We found statistical differences between the Harmony� and

Panorama� test results in reporting time (14.7 and 10.9 days,

respectively), mean fetal fraction (13.1 and 12.7%, respect-

ively) and MA. There were no differences regarding GA,

BMI, method of conception, percentage of conclusive results,

distribution of results (low risk, high risk and redraws) or low

fetal fraction cases (58%).

Fetal fraction

The average cfDNA fetal fraction was 12.7% (range 4.2–

27.9%) with no statistical differences for GA between 9.5 and

23.5 weeks. A low fetal fraction (58%) was found in 11.4% of

cases. Factors influencing cfDNA fraction were the type of

test (mean of 13.1% in the Harmony� test versus 12.7% in

the Panorama� test, p50.05), maternal weight (Spearman’s

2 C. Comas et al. J Matern Fetal Neonatal Med, Early Online: 1–6

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coefficient of �0.359, p50.01), BMI (Spearman’s coefficient

of �0.287, p50.01) and log b-hCG levels (Spearman’s

coefficient of 0.211, p50.01). We could not demonstrate any

relationship with GA, MA, PAPP-A levels, reason for referral

or fetal sex.

Discussion

Recently, analysis of cfDNA in maternal blood for NIPT has

been shown to be highly accurate in the detection of common

fetal autosomal trisomies. Since the commercial introduction

of NIPT for aneuploidies in clinical practice in developed

countries, several research studies and clinical experiences

have been published (Table 3). Clinical trials have demon-

strated the effectiveness of NIPT for main autosomal

trisomies in high-risk women [1–7,12,13,18,21,22]. More

recent studies in low-risk populations provide some, albeit

limited, evidence suggesting that cfDNA screening may be as

effective in the general population as it is in those already

scheduled for invasive testing [8,14–17,19,20]. As NIPT

has transitioned from clinical trials to clinical care, reports

are beginning to be published on laboratory screening

performance in clinical settings [17,20,25,26]. These reports

have demonstrated that, in a clinical setting, NIPT perform-

ance has met or exceeded characteristics established in prior

clinical validation studies. Our study summarizes the first

experience with NIPT in a private obstetrics practice in Spain.

We have described our initial 12-month experience with the

introduction of this new technology in private practice,

contributing to the expansion previously limited experience in

the general population, and showing that NIPT has the

potential to be a highly effective screening method as a

standard test for assessing the risk of major CA in a routine

clinical setting.

Two different approaches to NIPT are currently used in

clinical practice: counting methods, through whole-genome or

targeted sequencing, and SNP-based methods. Although there

is no conclusive evidence saying which technology is better

and we have the option of using both approaches, in our

center we offer a targeted non-counting method as a first

option. According to previously published experiences, this

technology offers some advantages, including greater clinical

coverage and sample-specific calculated accuracies. We

reserve targeted counting methods for egg donor pregnancies,

Table 2. Description of cases with ‘‘high risk’’ and ‘‘non-results’’ at NIPT.

CaseMA

(years)GA

(weeks)Fetal

fraction Indication FTS result NIPT result NIPT test Karyotyping

High-risk results at NIPT1 41 10.6 6.9 Advanced MA 1/2* High risk T21 Panorama� T212 33 17.1 11.7 High risk at FTS 1/199 High risk T21 Panorama� T213 43 14.4 7.0 Anxiety ** High risk 45.0 Harmony� 46,XX4 37 12.6 12.5 High risk at FTS 1/11 High risk T21 Panorama� T215 43 11.6 15.2 High risk at FTS 1/9 High risk T21 Panorama� T21

CaseMA

(years)GA

(weeks) Indication FTS result NIPT test Outcome

No results at NIPT1 40 18.1 Anxiety 1/1462 Harmony� Two samples; normal follow-up at delivery2 39 14.6 Advanced MA – Harmony� Redraw rejected, normal karyotype (amniocentesis);

normal follow-up at delivery3 39 11.4 Anxiety 1/1169 Panorama� Early vanishing twin normal follow-up at delivery4 41 15.1 Anxiety Low risk Panorama� Preeclampsia; IUGR

*FTS calculated a posteriori, **late booking, FTS not available. T21, trisomy 21.

Table 1. Maternal epidemiologic data and results of patients undergoing NIPT.

NIPT Harmony� Panorama� Overalln 120 213 333

MA (years) (mean, range) 37 (21–46)* 37 (29–46)* 37 (21–46)GA (weeks) (mean, range) 15.2 (9.5–23.5) 14.3 (10.6–23) 14.6 (9.5–23.5)BMI (mean, range) 22.9 (17.1–37.5) 22.9 (17.4–42.4) 22.9 (17.1–42.4)Conception (%) IVF 19 17 17.7Reporting time (days) (mean, range) 14.7 (10–21)* 10.9 (8–20)* 12.9 (8–21)Fetal fraction (%) (mean, range) 13.1 (5.9–22.7)* 12.7 (4.2–27.9)* 12.7 (4.2–27.9)Fetal fraction58%, n (%) 2 (11.8) 24 (11.4) 26 (11.4)Results n (%)

Conclusive results 117 (97.5) 207 (97.2) 324 (973)Low risk 116 (96.7) 203 (95.3) 319 (95.8)High risk 1 (0.8) 4 (1.9) 5 (1.5)No result 2 (1.7) 2 (0.9) 4 (1.2)Redraw 2 (1.7) 4 (1.9) 6 (1.8)False-positive rate 1 (0.9) 0 (0) 1 (0.3)

Bold values*:p50.05. n, number of cases; IVF, in vitro fertilization.

DOI: 10.3109/14767058.2014.947579 Non-invasive prenatal testing for major chromosomal anomalies in a clinical setting 3

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consanguinity families and twin pregnancies. Interestingly, in

our clinical practice we have not found any clinically

significant differences between Panorama� and Harmony�test results except in reporting time (lower for Panorama�).

In the near future, current cfDNA analysis turnaround time is

likely to be reduced, probably in both techniques, as further

technical advances are made. Fetal fraction distribution was

also different, with slightly lower levels for the Panorama�test, although with no clinical significance (the percentage of

fetal fraction lower than 8% was similar in both tests).

The study subjects were a combination of three different

populations. First were those found to be at high risk upon

FTS. As known, the major limitation of FTS is the relatively

high FPR. Therefore, NIPT would help identify these false

positives and avoid unnecessary invasive testing. Without

doubt, the main impact on clinical care has been the reduction

in prenatal invasive diagnostic procedures. Previous experi-

ences have demonstrated that the introduction of NIPT into

routine clinical settings could avoid 70–98% of invasive

procedures [15,17,27,28]. Similarly, in an empirical model,

according to our strategy, NIPT in patients deemed high risk

upon FTS could have reduced the number of invasive tests by

94% while maintaining the same detection rate for major

autosomal trisomies. However, in our clinical setting experi-

ence the real rate of decrease has only been 33%, which is

mostly due to the high cost of the test in our health care

system. Certainly, the main factor presently limiting wide-

spread application of the test is its high cost, which is

expected to decrease as technology advances and more

efficient methods are adopted.

The second group of women was those who tested negative

by conventional screenings but who were unable to alleviate

their anxiety. Interestingly, this is the most common reason for

referral in our experience (83.5%). Without NIPT many of

them would have chosen an invasive testing method, which,

from a risk-assessment point of view, tends to be unjustified.

The very low FPR of NIPT helped alleviate their anxiety,

without increasing their chances of requiring invasive testing.

For example, since the introduction of this technology in our

center invasive testing resulting from patient anxiety has been

reduced by 54% (based on our own unpublished data), which

is similar to previously published experiences [28]. This study

confirmed the very high specificity of NIPT.

The last group of patients was one that did not undergo any

prior screening, and were mostly referred for advanced MA.

For this group, NIPT detection rates and FPRs were

Table 3. NIPT for trisomy 21 in clinical trials.

ReferenceTechnological

approach Methodsn (T21 cases)

average GA (weeks) Results (%)

High-risk populationVerweij et al., 2013 t-MPS Prospective population study 520 (18) 14 S 94.4

Sp 100Nicolaides et al., 2013 SNPs Prospective population study 242 (25) 13.1 S 100

Sp 100Zimmermann et al., 2012 SNPs Retrospective population study 166 (11) 17 S 100

Sp 100Chiu et al., 2011 MPSS Prospective 753 (86) 13 S 100

Case control Sp 100Ehrich et al., 2011 MPSS Case control 480 (39) 16 S 100

Sp 99.7Palomaki et al., 2011, 2012 MPSS Case control 1742 (212) 15 S 98.6

Sp 99.8Bianchi et al., 2012 MPSS Case control 2882 (89) 15 S 98.9

Sp 100Sparks et al., 2012 t-MPS Case control 338 (72) 18 S 99.2

Sp 100Ashoor et al., 2012 t-MPS Case control 300 (50) 13 S 100

Sp 100Norton et al., 2012 t-MPS Prospective 3228 (81) 16 S 100

Sp 99.97Stumm et al., 2013 MPSS Prospective 522 (4421) 15.6 S 95.2

Sp 100Low-risk population

Lau et al., 2012 MPSS Prospective 567 (8) 14 S 100Sp 100

Nicolaides et al., 2012 t-MPS Retrospective 2049 (8) 12.6 S 100Sp 99.9

Dan et al., 2012 MPSS Prospective 11105 (143) 20 S 100Sp 99.96

Gil et al., 2013 t-MPS Prospective 1005 (11) 10 S 100Sp 99

Fairbrother et al., 2013 t-MPS Prospective 289 (no T21) 13 Successful result 98.6Song et al., 2013 MPSS Prospective 1916 (8) 16.5 S 100

Sp 99.94Bianchi et al., 2014 MPSS Prospective 2042 (5) 20 S 100

Sp 99.7

n, overall population; T21, trisomy 21 cases; S, sensitivity; Sp, specificity.

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significantly better than any other existing screening test and,

therefore, they chose this new technology as a primary option.

In our opinion, use of this test for population-based screening

requires further evaluation, particularly in terms of cost-

effectiveness. In fact, this is the way we counsel our patients.

However, in our private practice, after advising the patients

and assuming the costs of the test, few patients (2.4%)

maintained their decision in favor of this option.

Previous experiences have demonstrated the impact of fetal

fraction on the NIPT performance when screening for

aneuploidies [23]. Lower fetal fractions correlate with

increased error rates, particularly in counting methods [21].

Several co-variables for cfDNA fetal fraction have been

investigated, including reason for invasive prenatal diagnosis,

MA, screening marker levels and estimated aneuploidy risk.

According to our experience, fetal fraction was associated

with test type, maternal weight, BMI and maternal serum

b-hCG levels. Previous studies have shown a significant

negative correlation between fetal fraction and maternal

weight and a positive correlation with both PAPP-A and free

b-hCG levels [3,23,24,29]. Regarding GA, published data are

controversial. Wang et al. [24] found that fetal fraction

increased 0.1% per week from 10 to 21 weeks, while this

effect has not been found in other studies [3,6,29]. We did not

find any correlation with GA, probably due to the slight

increase in fetal fraction in this period in addition to the

limited sample size. Besides the aforementioned biologic

factors, many other unknown ones may be influencing cfDNA

fetal fraction. This lack of knowledge regarding the factors

involved warrants future research in this field.

One of the main concerns with NIPT is the incidence of

failed results as a consequence of insufficient fetal DNA or

other technical reasons. The failure rate may even be higher in

non-counting methods and in those resampled after an initial

failure [19]. With regard to the failure rate, our experience

shows similar figures compared to the previously published

series, with 2.7% of unsuccessful results, which is similar

between both counting and non-counting methods. In 83% of

the cases, a result was obtained with a second sample, a

proportion slightly higher than the 56% previously described

by Wang et al. [24] or the 68% described by Gil et al. [19].

This failure rate is relevant to comparisons with conventional

screenings, which rarely fail to obtain a result. However,

according to these experiences, about one-half to two-thirds

of test failures can be successfully resolved by drawing a

second sample later on in gestation. In our series, the success

rate was dependent on cfDNA fraction. On the contrary, we

did not find assay failure to be associated with BMI, PAPP-A

levels, GA or b-hCG levels.

False-positive rates for NIPT have been found to be

between 0.1 and 0.3% [17]. However, it remains unknown to

which extent discrepancies (between NIPT and fetal karyo-

types) are attributed to algorithmic deficiencies or to biologic

conditions yet to be clarified. Our series contributes a new case

of discordant Turner syndrome resulting from an unknown

biologic condition, with a targeted counting method, normal

amniotic fluid and maternal karyotypes. However, no follow-

up study of the placental cytogenetic features was performed.

As a result, the presence of confined placental mosaicism

cannot be entirely excluded. This type of discordance

undoubtedly requires further assessment, warrants difficult

counseling and definitively remains a challenge in this field.

The strengths of this study include its prospective design,

the collection of samples in a routine general population in a

real clinical setting, the appropriate gestational window, the

availability of complete clinical follow-up in up to 95% of

cases and the contribution of a new case of NIPT discordant

with Turner syndrome resulting from an unknown biologic

condition. In contrast to larger multicenter series where

follow-up was difficult to obtain [15], in our series 95% of

samples were validated, with no false negative cases;

therefore, we have a reliable insight of the true false negative

rate. The major limitation of this study was that the sample

size was relatively small to be able to reliably report detection

rates; however, four cases of DS were correctly detected,

expanding the previously published series of CA detected by

NIPT. In addition, we did not perform karyotyping in all cases

and so the assumption of euploidy was based on a lack of

phenotypic aneuploidy features in the newborns. This was an

inevitable consequence of the nature of the study, which was

based on a population undergoing routine screening for

aneuploidies, rather than on a high-risk population undergo-

ing invasive testing. Nevertheless, although confirmation of

the accuracy of results by phenotype analysis is not the

appropriate way in sex CA, it can be accepted for main

autosomal CA, which manifest phenotypically at birth.

Undoubtedly, the main impact of the introduction of NIPT

on clinical care has been a reduction in prenatal invasive

diagnostic procedures and risk-free reassurances for women

as a consequence of the lower FPR and higher positive

predictive value of this test compared with currently standard

screening programs [17]. Nevertheless, the commercial

introduction of NIPT raises several concerns. One major

issue is the optimal integration of NIPT into current screening

practices. Different strategies have been suggested. However,

it is still unclear which the most cost effective policy is. In the

absence of public health policies defining testing strategies,

the availability of professional guidelines, such as those from

the American College of Obstetricians and Gynecologists,

together with the Society for Maternal-Fetal Medicine [30],

can greatly facilitate this process by clarifying which

processes are scientifically valid and clinically acceptable

standards of care.

Acknowledgements

Under the auspices of the Catedra d’Investigacio en

Obstetrıcia i Ginecologıa del Departamento de Obstetricia,

Ginecologıa y Reproduccion del Hospital Universitario

Quiron Dexeus de la Universitat Autonoma de Barcelona.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

Carmina Comas is on the Speakers Bureau of Natera.

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6 C. Comas et al. J Matern Fetal Neonatal Med, Early Online: 1–6

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