Christin Coffeen, MS, LCGC

Senior Genetic Counselor

Illumina

Non-Invasive Prenatal Testing (NIPT):

Introduction and Technology Overview

© 2014 Illumina, Inc. All rights reserved.

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Birth Defects: Rates and Causes

Chromosomal (10–15%)

Normal (97%)
Prenatal Exposure (8–12%)

Defects (3%) Single Gene (2–10%)

Multifactorial (20–25%)

Unknown (40–60%)

Adapted from Stevenson, RE and Hall, J. Human Malformations and Related Anomalies, 2nd ed. 2006

2
Prenatal Prevalence of Reported
Chromosomal Abnormalities

Other rare
T21
16%
T18
Major fetal
aneuploidies
T13
Sex trisomy
5% 45,X
Sex trisomy
45,X T21
Other rare
8% 53%

T13
5%
T18
13%

Data adapted from Wellesley, D, et al., Rare chromosome abnormalities, prevalence and prenatal diagnosis rates
from population-based congenital anomaly registers in Europe. Eur J of Hum Gen 11 January 2012.

3
Prenatal Screening and Diagnostic Testing
Prior to NIPT
1st trimester 2nd trimester
Screen Screen
serum + U/S serum

1ST TRIMESTER 2ND TRIMESTER 3RD TRIMESTER

First day 40 wks

13 wks 27 wks
of LMP Term

CVS amnio
10-14 wks 16-22 wks

12 wks 18 wks

NT MEASUREMENT
(LIMITED ANATOMY)

4
Conventional Prenatal Screening Options
Detection Rates for Trisomy 21
Detection Rate (%)

1st Trimester NT Ultrasound 64-70

1st Trimester 1st Trimester Blood Screen NT Ultrasound 82-87

2nd Trimester Triple Screen 69

2nd Trimester Quadruple Screen 81

Integrated 1st Trimester

NT Ultrasound
2nd Trimester
94-96
Screen Blood Screen Blood Screen

Serum 2nd Trimester Blood

1st Trimester Blood Screen 85-88
Integrated Screen

ACOG Practice Bulletin No. 77, January 2007

False Positive Rate: 5%
Actual detection rates and false positive rates will
vary slightly based on the laboratory used.

5
Invasive Prenatal Testing

Gold-standard diagnostic tests

– Chorionic Villus Sampling (CVS) at 11-13
weeks
– Amniocentesis at 15-20 weeks
Present risk to patient and fetus
– 0.4% risk of miscarriage with amniocentesis*
– Risk of maternal bleeding, infection, leaking

*Ultraschall Med. 2012 Dec;33(7):E75-9. doi: 10.1055/s-0031-1299388

6
Ultrasound Examination

Useful because fetuses affected with aneuploidy often have

anatomic changes or anomalies.
A genetic sonogram uses ultrasound to assess the fetus for both
structural anomalies and soft markers suggestive of aneuploidy
Invasive testing still is required to obtain a definitive diagnosis.
First and Second trimester ultrasound
– Can detect many other abnormalities that can be associated with
other chromosomal/genetic syndromes
– Can detect structural abnormalities not associated with genetic
syndrome

7
What are the Goals of NIPT?

Reduce Reduce
exposure of false
risk to fetus positives

Testing that Enable a

can easily high
be offered to detection
pregnant rate
women

8
 NIPT Technology Overview

9
Non-Invasive Prenatal Testing (NIPT)
A new category of prenatal testing
Detect fetal aneuploidy using cell-free DNA from maternal
blood
– Analyzed by next-gen DNA sequencing

Other used nomenclature:

– NIPD: Noninvasive Prenatal Diagnosis
– NIPS: Noninvasive Prenatal Screening
– cfDNA: Cell-free DNA
– cffDNA: Cell-free fetal DNA
– DNA-based noninvasive prenatal screening

10
Cell-Free DNA (cfDNA)
A reliable analyte during pregnancy

Released through apoptosis

– Fetal cfDNA likely arises from
cytotrophoblastic cells of placenta

Released into bloodstream as small DNA

fragments (150–200 bp)
Maternal blood contains both fetal,
maternal cfDNA
– 2–20% of total cfDNA is fetal

Fetal cfDNA reliably detected after 7+

weeks gestation
Fetal cfDNA undetectable within hours
postpartum
1. Barrett, A. et al. Implementing prenatal diagnosis based on cell-free fetal DNA: Accurate identification of factors affecting fetal DNA yield.
PLoS One 6; (2011);e25202.
2. Nigam, A. et al. Detection of fetal nucleic acid in maternal plasma: A novel noninvasive prenatal diagnostic technique. JIMSA 25:119–
200(2012).

11
Massively Parallel Sequencing (MPS)

Fetal DNA
fragments in
maternal blood.

Cell free DNA

fragments are
then sequenced.

Compare the
individual
sequenced
chromosomes
against a reference
for analysis.

12
Genome-Wide MPS
Provides precise, across-the-genome coverage
NOT TO SCALE

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20

21 22 X Y Chromosome-Wide Coverage

Benefits
– Low assay failure rates
– Ability to add new content to test menu

13
Targeted MPS
Limited to few chromosomes, loci
NOT TO SCALE

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20

21 22 X Y Chromosome-Wide Coverage

Drawbacks
– High assay failure rates
– Limited ability to add new content without changing assay

14
Targeted MPS (SNP-Based Method)
Complex, failure-prone method

SNP
Sequencing

Plasma = Maternal +
Maternal + Fetal
Fetal cfDNA Genotype
Deduce
Maternal Risk
Fetal
Blood Result
Genotype
Buffy Coat = Maternal
Maternal DNA Genotype

Drawbacks
– High Assay Failure Rates
– Difficult to analyze egg-donation, surrogacy, consanguinity,
maternal transplant, multiple gestation samples

15
Evidence for NIPT Performance with MPS
Updated Meta-analysis: To review the clinical validation of cfDNA screening for
fetal aneuploidies

37 publications on NIPT for detection of aneuploidies between 2011-2015

DR (%) 95% CI FPR (%) 95% CI

Trisomy 21 99.2 98.5-99.6 0.09 0.05-0.14
Trisomy 18 96.3 94.3-97.9 0.13 0.07-0.20
Trisomy 13 91.0 85.0-95.6 0.13 0.05-0.26
Monosomy X 90.3 85.7-94.2 0.23 0.14-0.34
Other sex 93.0 85.8-97.8 0.14 0.06-0.24
aneuploidies

Twins T21 93.7 83.6-99.2 0.23 0.00-0.92

Gil et al (2015). Ultrasound Obstet Gynecol, 45: 249-266

16
NIPT with Arrays?
An Unknown Limit of Detection

Drawbacks
Array NIPT unproven
High samples failure
Likely requires high FF call
Late term
High FF means late term testing
Juneau et al (2014). Fetal Diagn Ther, DOI: 10.1159/000367626.
Stokowski R, Wang E, White K, et al (2015) Prenat Diagn, DOI: 10.1002/pd.4686

17
 Proof is in the Data

Published and presented samples1

500.000
400.000 382.996

300.000
200.000
100.000 37.206 32.916
878
-
WGS Targeted Array Targeted SNP
Sequencing Technology Sequencing

1 A PubMed search for “cell-free, DNA, prenatal”, “noninvasive prenatal testing”, and “noninvasive prenatal screening” was performed on April
30, 2015. All validation and clinical studies using unique samples were included, where a current clinical NIPT provider performed sample
analysis. Case studies and studies published in a language other than English were excluded. Data from a 2015 ESHG conference abstract was
also included. A total of 45 published studies were surveyed. Data calculations on file. Illumina, Inc. 2015. NGS = next-generation sequencing;
either whole-genome or targeted.

18
Illumina Technology Has Enabled NIPT

Company Approach Sequencing Platform

Whole Genome

Whole Genome

Targeted SNP Sequencing

Whole Genome

Whole Genome

Illumina NGS platform clinically validated for NIPT on over 35,000 patients;
over 1,000,000 clinical reports issued

19
 Clinical Implementation and Counseling
Considerations

20
Professional Society Guidelines Endorsements

21
 ISPD Position Statement April 2013

 “Non-invasive prenatal testing based on massively parallel sequencing of circulating free

fetal DNA (cfDNA) in maternal plasma has been shown to be highly effective for
aneuploidy detection”
 “[NIPT] would appear to be the most effective method for screening for fetal trisomy 21
and trisomy 18”
 “The tests should not be considered to be fully diagnostic and therefore are not a
replacement for amniocentesis and CVS”
 “Laboratory providers should also be prepared to provide ongoing specifics on accuracy, test
failure rates and turn-around time”
Also supporting NIPT for high risk pregnancies:

22
ISPD Position Statement on Chromosome
Abnormality Screening: April 2015

Some important points from position statement:

– cfDNA screening as a primary test offered to all pregnant
women
– cfDNA secondary to a high risk assessment based on serum
and ultrasound screening
– cfDNA contingently offered to a broader group of women
ascertained as having high or intermediate risks by
conventional screening
 Contingent provision of cfDNA, could also include a protocol in
which women with very high risks are offered invasive prenatal
diagnosis while those with intermediate risk are offered cfDNA

23
Which Patients Should Be Offered NIPT?

Patients wanting early, accurate testing and are at high risk of

aneuploidy due to:
Maternal age-related risks
Positive results on maternal-serum screening
Abnormal ultrasound finding(s)
History suggestive of increased risk for T21, T18,T13 or sex
chromosome aneuploidy
Parental translocation involving one of the tested chromosomes

Patients wanting early, accurate testing and are at average risk

of aneuploidy

24
 NIPT Failure Rates by Technology & Company

7%
6% 6,4%
5%
4%
3%
3,0%
2%
1% 1,3%
0.1%
0%
verifi1 MaterniT212 *Harmony3 Panorama4
Whole Genome Targeted Sequencing
Sequencing
1Taneja et al. Abstract presented at ESHG, 2015
2McCullough RM et al. PLoS One. 2014 *Very limited data published using array
3Norton ME, et al. New Engl J Med 2015
4Dar, et al. Am J Obstet Gynecol. 2014
technology, no clinical experience available

25
Why Do Test Failures Matter in NIPT?

Actual sensitivity is less than claimed

Up to
sensitivity

Studies have shown a high rate of

22%
aneuploidy in test failures Aneuploidy
Rate1,2
Redraw for NIPT is usually ineffective in NIPT test failures

– High published redraw failure rates

– Leads to increased turnaround time, office Up to
visits, patient/physician frustration
65%
NIPT Redraw
Failure
Rate1,2
1 Pergament E, et al. Obstet Gynecol. 2014 Aug;123(2 Pt 1):210–8
2 ACOG Committee Opinion Number 640, Sept 2015

26
 Women whose results are not reported, indeterminate, or
uninterpretable (a “no call” test result) from cell-free DNA
screening should receive genetic counseling and be offered
comprehensive ultrasound evaluation and diagnostic testing
because of an increased risk of aneuploidy.

27
Key Points to Remember
NIPT is now part of prenatal screening options.
Important to remember the benefits and limitations of the various
prenatal tests
– NIPT does not test for all chromosome abnormalities, birth defects, genetic
disorders or other pregnancy complications.
– No testing is 100%.
Labs have varying restrictions regarding gestational age, multiples,
consanguinity and pregnancies conceived through the use of donor
eggs/surrogacy.
Labs have varying test failure rates (some of which may include an
increased risk of aneuploidy).
Possibility test results might not reflect the chromosomes of the fetus,
but may reflect chromosomal changes to the placenta or of the mother.
Co-twin demise

28
 Thank You

29
 Appendix

30
 Whole Genome Sequencing Has Benefits Over
Targeted Sequencing & Arrays

WGS provides precise counts, Targeted sequencing is limited

across the genome to few chromosomes, loci
Benefits Drawbacks
• Low assay failure rates (<1%) • High assay failure rates (up to 12%)
• Ability to add new content to test menu • Limited ability to add new content without changing assay

31
 Fetal Fraction in NIPT
Fetal Fraction = amount of fetal cfDNA in total cfDNA

% fetal fraction (FF) affects ability of NIPT to detect

fetal aneuploidy
– Very low fetal fraction may lead to false negative results

Several methods currently in use to estimate fetal

fraction
– Inaccurate at low fetal fraction (much variation in the
measurement)
Threshold for fetal fraction depends on coefficient of
variation obtained for an individual chromosome
– May be improved through algorithm improvements

32
Finding the Fetal Fraction

Assay Quality
– Lowers the limit of detection (LOD)
– Based on sequencing methodology and analysis method
Fetal Fraction
– Lower fetal fraction demands a lower LOD

Assay Quality

Limit of Detection

Fetal Fraction

33
Why does anyone measure Fetal Fraction?

NIPT assays with lower quality use Fetal Fraction to eliminate

difficult samples
– Eliminating samples with low fetal fraction increase sensitivity and
specificity
Some labs do not measure fetal fraction and do not eliminate
samples from analysis
Laboratory Clinical experiences
– Assay failure rates
– Negative Predictive Value (NPV)
Lowest limit of detection
– Combination of accurate sequencing and data analysis algorithms

34
 Clinical Factors Affecting Fetal Fraction
Significant correlation with aneuploidy
– FF higher for trisomy 21
– FF lower for trisomy 18, trisomy 13, monosomy X

Correlation with gestational age

– Slight increase from 10-21 weeks gestation
– Significant increase after 21 weeks gestation

Weak correlation with maternal BMI

– Slight decrease in FF with maternal BMI
– No specific threshold has been established where results
cannot be obtained relative to maternal weight
Not affected by maternal age, ethnicity, a priori trisomy
risk
Rava et. al, Clin Chem 2013 Jan;60(1):243-50.; Wang, E. et al. Prenat Diagn 2013 Jul;33(7):662-6.
Galbiatia, D. et al. Hum Genet 2005,117(2-3):243-8.; Bianchi, D., et al. N Engl J Med 2014 Feb 27;370(9):799-808.

35
Fetal Fraction in NIPT
Vast majority of samples have FF levels well above lower threshold

From Wang et al. Prenatal Diagnosis 2013 33, 1-5.

36
Comparison of NIPT Service Providers
iIlumina verifi® prenatal test leads in performance
Illumina MaterniT21 Harmony Panorama
verifi Sequenom Ariosa Natera

Method Whole Genome Whole Genome Targeted/Array Targeted

4%5
Limit of Detection 1.4–2.71 4%3 (unknown with 3.8–8.08
microarray)
2 tubes maternal
2 tubes maternal 2 tubes maternal
Specimen 1 tube maternal blood
blood blood
blood, paternal
sample optional
4.6–4.9%5,6
Failure Rate 0.1%2 1.9%4 (unknown with 6.4–8.1%8,9
microarray)
3–5 5 7–10 9.29
Time to Report business days business days business days calendar days

Egg Donors Yes

Yes Yes No
& Twins (13% failure rate7)

Microdeletions
Yes Yes No Yes
Offered
1. Rava, et al., Clin Chem 2014;60(1):243-50
2. Bhatt et al. Poster presented at ISPD, 2014
• Rabinowitz,
3. Jensen TJ, et al.PLoS
et al. ASHG ONEAbstract
2013:8(3):2012.;
e57381.Presented data at NSGC AEC 2012
doi:10.1371/journal.pone.0057381
• Norton
4. ME, etRM,
McCullough al. etAm
al., JPLoS
Obstet
ONEGynecol. 2012doi:10.1371/journal.pone.0109173
9(10): e109173. doi:10.1016/j.ajog.2012.05.021
• 5. Norton ME,
Palomaki GE,et et
al. al.
AmGenet
J ObstetMed.
Gynecol.
20122012 doi:10.1016/j.ajog.2012.05.021
Mar;14(3):296-305; M. Ehrich communication
6. Nicolaides, et al., Am J Obstet Gynecol 2012;207(5):374.e1-6
• Futch et al., Prenat Diagn 2013 Apr [Epub ahead of print]
7. Gil, et al., Fetal Diagn Ther 2013
8. Pergament E, et al. Obstet Gynecol. 2014 Aug;123(2 Pt 1):210-8
37 9. Dar P, et al., Am J Obstet Gynecol (2014), doi: 10.1016/j.ajog.2014.08.006.
Comparison of NIPT Service Providers
illumina verifi Prenatal Test Leads in Performance

Illumina NIFTY
verifi BGI

Method Whole Genome Whole Genome

Failure Rate 0.1% ~2% before redraw1,3

3–5 10–15
Turn Around Time
business days business days2,3,4

Sample (blood) 1 tube maternal blood 2 tubes maternal

Published Laboratory
Yes Yes
Clinical Experience

CLIA/CAP-certified Non CLIA/CAP-certified

Laboratory
laboratory laboratory

1. Zhang et al Ultrasound Obstet Gynecol. 2015 Jan 19. doi: 10.1002/uog.14792.

2. http://www.niftytest.com/wp-content/uploads/2014/09/BGIDX_NIFTY_Leaflet_24.06.2014_New_Code.pdf
3. http://www.thisismy.co.uk/non-invasive-prenatal-testing-nipt/
4. http://igenescreen.com/for-doctors/

38
NIPT Test Failure Rates

Failure rate in twins

With paternal sample 13.2%

Requires retest of sample
NIPT Failure Rate

8,1%

5,5%
~5.0% 4,6%

1.9%
0.1%

7
Natera1,2 Ariosa3,4 Sequenom5,6

Targeted Sequencing Whole Genome Sequencing

1. Pergament E, et al. Obstet Gynecol. 2014 Aug;123(2 Pt 1):210-8
2. Dar P, et al. Am J Obstet Gynecol. 2014 Aug 8. doi: 10.1016/j.ajog.2014.08.006
3. Norton ME, et al. Am J Obstet Gynecol. 2012 doi:10.1016/j.ajog.2012.05.021
4. Gil M, et al. Fetal Diagn Ther. 2014;35(3):204-11
5. Palomaki GE, et al. Genet Med. 2012 Mar;14(3):296-305
6. McCullough RM, et al., PLoS ONE 9(10): e109173. doi:10.1371/journal.pone.0109173
7. Bhatt et al. Poster presented at ISPD, 2014
39
Genetic Counseling NIPT Flipbook

10 pages with illustrations to

help patients understand the
NIPT testing process,
conditions tested, result
interpretation
Used by healthcare providers
prior to NIPT
verifi prenatal test specific
Available in 9 languages:
English, Spanish, Portuguese,
French, German, Italian,
Korean, Japanese, Chinese
Download at verifitest.com
Tools For Your Practice

40
Patient Education Video

12-minute video providing an overview of the benefits and limitations of various

prenatal testing options
– Prenatal screening (e.g. first trimester combined screen)
– CVS/Amniocentesis
– NIPT (verifi prenatal test specific)
 Includes description of conditions tested

Healthcare providers can direct their patients to watch

this video in the clinic or at home prior to their
OB appointment
Available in 9 languages: English, Spanish,
Portuguese, French, German, Italian, Korean,
Japanese, Chinese
Can be viewed at verifitest.com
Tools for Your Practice
– Downloadable to other Practices’ websites

41
NIPT Test Failures Not Only Due to Fetal Fraction Cutoffs
Comparison of test providers
Other Technical Failures*
8.1% Fetal Fraction
Test Failure Rate

2,0%
4.6%

6,1% 2,8% 1.9%

1,0%
1,8% 0,9%
0.1%
1 2 4
Natera Ariosa Sequenom 3 verifi

LOD: 3.8–8.0%1 4%2 4%3 1.4–2.7%5

Test Failure Rates Depend on Assay Reliability, Limit of Detection (LOD)

* Reasons include insufficient cfDNA, inability to measure fetal fraction, lab error, contamination, bad statistical fit,
highly variable cfDNA counts, or other sequencing failure
1. Pergament E, et al. Obstet Gynecol. 2014 Aug;123(2 Pt 1):210-8
2. Norton ME, et al. Am J Obstet Gynecol. 2012 doi:10.1016/j.ajog.2012.05.021
3. McCullough RM, et al., PLoS ONE 9(10): e109173. doi:10.1371/journal.pone.0109173
4. Bhatt et al. Poster presented at ISPD, 2014
5. Rava, et al., Clin Chem 2014;60(1):243-50

42
Implications of Test Failure
Theoretical population 100,000 Pregnancies, T21 Prevalence 1:500

T21 (n=200) Normal (n=99,800)

Screening Detection Cases False Failure Invasive Procedure

Method Rate Detected Positive Rate Tests Related
Rate Loss

Maternal age 30% 60 5% - 4,990 10

Integrated Screen 95% 190 5% - 4,990 10

Natera >99.9% >199 <0.1% 6.3% 6,287 12

Ariosa >99.9% >199 <0.1% 3% 3,094 6

Sequenom 98.6% 197 <0.1% 1.9% 1,996 4

Verifi >99.9% >199 <0.1% 0.1% <100 <1

43
 NIPT as a Primary Screen
START HERE

CVS/Amniocentesis
NIPT
(Invasive)

• Maternal age-related risks

• Abnormal ultrasound Aneuploidy YES

detected or
finding(s) suspected?
(Genetic counseling is recommended)

• Hx suggestive of increased NO

risk for T21, T18,T13 or SCA

Continue with pregnancy Continue with pregnancy
management according to your management according to your
practice’s protocols practice’s protocols
• Parental translocation
involving one of the tested
chromosomes

44
NIPT as a Secondary Screen
(following a positive serum screen)
(START HERE)

Serum Screening
(per practice’s protocol)

Is the serum YES Counsel the patient about the

CVS/Amniocentesis
screen result verifi prenatal test and invasive NIPT
positive? test options (Invasive)

NO

Aneuploidy YES
detected or
suspected?
(Genetic counseling is recommended)

NO
Continue with pregnancy
management according to your
practice’s protocols

45
Clinical Methods in Published Series

Current Clinical No. of Published

Test (Company)
NIPT Method NIPT Samples
Bambni™ Assay (Berry Genomics) Illumina NGS 2,351
MaterniT21 PLUS™ Test (Sequenom) Illumina NGS 108,665
NIFTY™ Test (BGI) Illumina NGS 160,667
Panorama™ Prenatal Screen (Natera) Illumina NGS 32,916
PrenaTest (LifeCodexx AG/GATC Biotech
Illumina NGS 504
AG)
verifi® Prenatal Test (Illumina) Illumina NGS 113,367
Harmony™ Prenatal Test (Ariosa) Illumina NGS 37,206
Harmony™ Prenatal Test (Ariosa) Affymetrix Array 878
A survey of 45 published studies* revealed that 99.8% of reported NIPT samples
run on Illumina NGS systems
* A Pubmed search for “cell-free, DNA, prenatal”, “noninvasive prenatal testing”, and “noninvasive prenatal screening” was performed on April 30, 2015.
All validation and clinical studies using unique samples were included, where sample analysis was performed by a current clinical NIPT provider.
Case studies and studies published in a language other than English were excluded. Also included data from a 2015 ESHG conference abstract.

46