1. Non-Invasive Prenatal Testing (NIPT)
Developed by Dr. June Carroll, Ms. Shawna Morrison and Dr. Judith Allanson
Last updated April 2015

2. Disclaimer
This presentation is for educational purposes only and should not be used as a substitute for clinical judgement. GEC-KO aims to aid the practicing clinician by providing informed opinions regarding genetic services that have been developed in a rigorous and evidence-based manner. Physicians must use their own clinical judgement in addition to published articles and the information presented herein. GEC-KO assumes no responsibility or liability resulting from the use of information contained herein.

3. Objectives Following this session the learner will be able to:
Refer to their local genetics centre and/or order genetic testing appropriately for non-invasive prenatal testing (NIPT)
Discuss and address patient concerns regarding NIPT
Find high quality genomics educational resources appropriate for primary care

4. Typical Prenatal Testing Algorithm
Offer PN screening to all pregnant women
FTS/IPS/SIPS
NIPT for AMA and for women willing to pay
Family history
Ethnicity-based screening
If negative or decline
If positive
*for ethnicity-based screening, if both members of the couple are carriers of the same condition
18-20 week fetal morphology scan
Refer to Genetics
If indicated (e.g. fetal anomalies )

5. Prenatal Testing Algorithm for Women at Increased Risk
Indication
Advanced maternal age, multiple soft markers on ultrasound, ultrasound anomaly, positive prenatal screen, etc.
Genetic counselling with testing options
No further testing
Invasive Testing
(diagnostic)
Screening Test
e.g. NIPT
If positive
If negative
QF-PCR
Detects common aneuploidies: Down syndrome, Trisomy 18, Trisomy 13 and sex chromosome aneuploidies
Depending on indication:
No further testing
Consider additional testing
2.4% CMA will pick up abnormality for any indication
6.4% if u/s abnormality
~1% VUS
If positive
Karyotype
If negative
No further testing
Additional Testing
e.g. Chromosomal microarray

6. Case 1
A 32 year old woman has had a positive Integrated Prenatal Screening Test (IPS) result for Down syndrome. She is about 17 weeks gestation.
Is NIPT a good option?

7. Case 2
A 40 year old G1 woman is about 9 weeks gestation. She is in your office to discuss prenatal testing options in this pregnancy conceived by IVF.
Is NIPT a good option?

8. Case 3
A 29 year old patient had a nuchal translucency (NT) of 4.4mm at 12+5 weeks gestation
You offered NIPT and she accepted
NIPT results were normal. She is now 14+2 weeks gestation
What are the next steps?

9. What is Non-Invasive Prenatal Testing?
Screening test to prenatally detect Down syndrome and other aneuploidies (extra or missing chromosomes)
trisomy 21, 18, 13
trisomy of sex chromosomes (XXX, XXY, XYY)
Turner syndrome (monosomy X)
triploidy (extra copy of all chromosomes)
Aneuploidy: refers to an abnormal chromosome number (extra or missing)

10. What is Non-Invasive Prenatal Testing?
NIPT measures circulating cell-free DNA (cfDNA) from placenta present in maternal blood
‘fetal’ cfDNA comprises ~10% of DNA in maternal blood
Increases with gestational age
Companies offering NIPT use various technologies to analyze cfDNA and determine chromosome quantities
Performed on maternal blood sample
As early as 9 weeks gestation (company specific)
Prior U/S preferable– viability, accurate GA, exclude multiples
Technologies:
detect higher relative amounts of DNA from an aneuploidy fetus by comparing quantity to a reference chromosome, determining if there is a normal, higher or lower than expected quantity of particular DNA sequences
amplify and sequence chromosomes of interest using single-nucleotide polymorphisms (SNPs)

11. How does non-invasive prenatal testing compare to traditional prenatal screening for Down syndrome?
Screening test
Test info
Detection rate / Sensitivity for T211
False Positive Rate for T211
Positive predictive value for T212,3
FTS
MA, NT, PAPP-A, beta-hCG
80-85%
3-9%
~4%
IPS
MA, NT, PAPP-A, AFP, uE3, hCG
85-90%
2-4%
Quad/MSS
MA, AFP, uE3, total hCG, inhibin
75-85%
5-10%
SIPS
MA, PAPP-A, AFP, beta-hCG/total hCG, uE3, inhibin
80-90%
2-7%
NIPT
+/-MA, cfDNA
>99% %
~80.9% for all populations (high and low risk women)
Norton 2015 – PPV for NIPT for T21 was 50% in low risk women and 76.0% in women <35y
Recommendations: any prenatal screen offered to women who present in T1 should have DR of 75% and no more than 3% FPR, if presenting in T2 DR of 75% with no more than 5% FPR
Open Neural Tube Defect (ONTD): Maternal Serum – alphafetoprotein (MSAFP) in T2 + comprehensive U/S screen at weeks
U/S for delayed ossification of fetal nasal bone – T1 (11-14 weeks) or T2
T1 DR 69% FPR depending on maternal ethnicity (9% Afro-Caribbeans, 5% Asians, 2.2% Caucasian)
1 Prenatal Screening Ontario
2 Bianchi et al 2014 N Engl J Med 370:9
3 Norton et al 2015 N Engl J Med Apr 1 [Epub ahead of print]

12. Non-Invasive Prenatal Testing (NIPT) Landscape
Increasing demand from women
Increasing uptake in most (urban) centres
3 separate companies, 3 separate technologies
Costs between $795 and $1200
8-10 days for result

13. Recommendations Offer to all women:
Prenatal screening using either FTS, IPS or MSS (SIPS or Quad)
Fetal morphology scan at about weeks gestation
Consider NIPT as an option for women who have a high risk for having a baby with an aneuploidy
Offer to everyone?
Society of Obstetricians and Gynaecologists of Canada, American College of Obstetricians and Gynecologists, International Society for Prenatal Diagnosis, National Society of Genetic Counselors, Society for Maternal-Fetal Medicine

14. Consider offering Non-Invasive Prenatal Testing (NIPT) for women who:
Are of advanced maternal age, defined as 40 years of age or older at estimated date of birth
Have an abnormal multiple marker screen i.e. FTS/IPS/MSS
Have a fetal nuchal translucency (NT) measurement of 3.5mm or greater 
Have had a previous pregnancy or child with aneuploidy

15. Consider offering Non-Invasive Prenatal Testing (NIPT) for women who:
Have other high risk factors:
Fetal congenital anomalies on ultrasound highly suggestive of trisomy 13, 18 or 21*
Soft markers on ultrasound which are highly suggestive of aneuploidy [Refer to SOGC guidelines, 2005].*
Are at risk of carrying a male fetus with an X-linked condition (NIPT would be used for sex determination)*
In Ontario, MOHLTC funding applications for the indications above marked by an asterisk (*) must be submitted by a geneticist or maternal fetal medicine (MFM) specialist. In other provinces circumstances may be different. Consult your local genetics centre or MFM specialist.

16. Non-Invasive Prenatal Testing (NIPT) results
Results will be reported in various ways and may be worded as:
positive or negative
aneuploidy detected, no aneuploidy detected or aneuploidy suspected/borderline value
high risk or low risk

17. Non-Invasive Prenatal Testing (NIPT) results
If the result is negative, this is reassuring
Your patient should still be offered:
fetal morphology scan at weeks’ gestation
referral for genetic and/or maternal fetal medicine consultation, which may be indicated for additional counselling and testing, depending on the reason your patient qualified for NIPT (e.g. increased NT)
As per SOGC guidelines, MS-AFP should only be offered to pregnant women with a pre-pregnant body mass index ≥ 35 kg/m2 or when geographical or clinical access factors limit timely and good quality ultrasound screening
SOGC - Society of Obstetricians and Gynaecologists of Canada

18. Non-Invasive Prenatal Testing (NIPT) results
If the result is positive:
Genetic counselling
Confirmation by diagnostic testing
No irrevocable obstetrical decisions should be made in pregnancies with abnormal NIPT results without confirmatory invasive testing (CVS or amniocentesis) - SOGC

19. Benefits of Non-Invasive Prenatal Testing (NIPT)
Fewer women having diagnostic tests with associated risk of pregnancy loss
Early test result (drawn at ≥ 9-10 weeks at earliest)
No risk of miscarriage
Detects the most common chromosomal aneuploidies
Higher detection rates and lower false positive rates than IPS or MSS

20. Limitations of Non-Invasive Prenatal Testing (NIPT)
NIPT cannot:
Detect chromosome differences other than aneuploidy of chromosomes 13, 18, 21, X and Y
some companies are now adding screening for other trisomies and certain microdeletion syndromes
Completely rule out aneuploidy
Detect single gene conditions
Detect congenital anomalies
Failed results (~6%)
1/2- 2/3 can be successfully resolved with redraw at later gestation
False positives and false negatives
Twins and IVF pregnancies
No result: . According to recent review (Cuckle, 2015) There are three circumstances when no result can happen: (1) a poor sample or quality assessment failure; (2) a low FF; or (3) an ‘uninterpretable’ result, regarded by the laboratory as too close to the cut-off 
? About reason for FPR can be addressed in Q&A – low ff, confined placental mosaicism, maternal aneuploidy, vanishing twin
Twins- lower sensitivity in discordant twins – lower ff per twin
IVF – limited data, may have higher fail rate
Lambert-Messerlian et al., (2014) have compared the performance of NIPT for autosomal aneuploidy in pregnancies achieved by assisted reproductive technologies (ART) versus controls. There was no significant difference in cell-free DNA fetal fraction between the two groups. However, they did find that z-scores for trisomy 21 and trisomy 18 were lower in the ART group. They conclude that the findings need to be confirmed before any adjustment is made to testing and reporting protocols. It is also possible that ART pregnancies are more likely to include a vanishing twin and residual placental tissue. If these findings are confirmed, it may be useful to make minor adjustments to the z-scores or their interpretation in pregnancies conceived via ART.

21. Non-Invasive Prenatal Testing: Discordant results
Condition
Overall
True Positive
Overall False Positive
Down syndrome
91-93%
6-9%
Trisomy 18
60-77%
23-47%
Trisomy 13
44-54%
46-56%
Sex chromosome abnormality (varies with type e.g. Monosomy X, XXY, XYY)
38-88%
12-100%
Table 2 Wang – 3 studies
The most common initial NIPT-positive result was trisomy 21 (41 cases), followed by trisomy 18 (25 cases), trisomy 13 (16 cases), sex chromosome aneuploidy (16 cases), trisomy 16 (3 cases), monosomy 21 (2 cases), and 1 case each of triploidy and microdeletion of 22q11.2. Four samples negative for NIPT but positive for ultrasound findings were included.
Cheung
Monosomy X True POS 8/21 (38) Tru NEG 13/21 (62)
XXX or XXY /17 (88) /17 (12)
XYY / /1 (100)
The positive predictive value (PPV) is proportional to the specificity (true negative) of the test and the prevalence of the disorder
Wang et al Genet in Med 17(3)
Cheung et al 2015 N Engl J Med [Epub ahead of print]

22. Limitations of Non-Invasive Prenatal Testing: Biological factors
Vanishing twin
Fetal fraction (ff)
Mosaicism
Maternal BMI  ff at 11-13W in 60kg woman = 11.7% and in 160kg woman 3.9%

23. Limitations of Non-Invasive Prenatal Testing: Biological factors
Fetal fraction (ff)
Is the amount of fetal cfDNA divided by total (maternal and fetal) cfDNA
Low ff result in test failures (<4%)
Is affected by:
Maternal BMI
Gestational age
Multiple gestations
Higher overall ff, but lower per fetus, risk of false negative
Mosaicism
High level of mosaicism vs low level mosaicism
Maternal BMI  ff at 11-13W in 60kg woman = 11.7% and in 160kg woman 3.9%

24. Limitations of Non-Invasive Prenatal Testing: Biological factors
Confined placental mosaicism
Present in 1-2% of first trimester placentas
Trisomy 13 and 18  lower placenta volume
Rava RP et al. (2014). Clinical Chemistry, 60,
Wegrzyn et al. (2005). Ultrasound in Obstetrics & Gynecology, 26,

25. Limitations of Non-Invasive Prenatal Testing: Control and Standards
Takoudes and Hamar (2014) made headlines when blood samples from 2 non-pregnant women were sent to 5 American commercial labs offering NIPT
3/5 labs reported normal result (no aneuploidy)
2/5 labs unable to report due to low fetal fraction
Some companies differ on their assertions that ff influences accuracy of NIPT testing

26. Non-Invasive Prenatal Testing (NIPT) and counselling
Pre- and post-test counselling is important
Invasive testing following positive results
Conditions tested for in addition to T21
Incidental findings
Consult genetics if unsure
Geneticseducation.ca has more information, links to companies and sample completed forms
Refer for genetic counselling when appropriate
Company websites:
Harmony Prenatal Test™ by Ariosa Diagnostics through Gamma-Dynacare
Panorama™ by Natera through Lifelabs/CML
Verifi® Prenatal Test by Verinata through Mount Sinai Services Inc. or Medcan Clinic
Incidentals e.g. maternal aneuploidy, maternal malignancy
Other companies offering NIPT:
Sequenom
Counsyl
Nukleo in Quebec

27. Ordering Non-Invasive Prenatal Testing (NIPT) in Ontario
Educational Resources > GECKO on the run > NIPT

28. Expansion of non-invasive prenatal testing
In October 2013 one company expanded their NIPT test to include screening for microdeletion syndromes (e.g. 22q deletion/DiGeorge and trisomies 16 and 22), and in spring 2014 others followed suit
Rationale:
Cumulatively common disorders
The combined at-birth incidence of the 5 commonly offered microdeletion syndromes is approximately 1 in 1,000
Incidence is independent of maternal age
Appear to have high sensitivity BUT no clinical validation studies, no guidelines, rare conditions decrease PPV and increase FPR
No MOHLTC approval
Incidence = 1 in 4,000 to 1 in 50,000

29. Case 1
A 36 year old woman has had a positive Integrated Prenatal Screening Test (IPS) result for Down syndrome. She is about 17 weeks gestation.
Is NIPT a good option?

30. Yes But consider: She is eligible for NIPT with MOH funding
In the event of an abnormal result, is termination of pregnancy an option for the couple?
More rapid result from amniocentesis, consider GA
If NIPT is positive, guidelines recommend confirmatory diagnostic testing by amniocentesis – delays timing for diagnosis
What is her IPS risk?
1 in 2 versus 1 in 120

31. Case 2
A 40 year old G1 woman is about 9 weeks gestation. She is in your office to discuss prenatal testing options in this pregnancy conceived by IVF.
Is NIPT a good option?

32. Yes
Advanced maternal age (greater than 40 years at EDB) is an appropriate indication for NIPT
Covered by MOH funding
Better screen than IPS
Earlier result
Decreased chance with NIPT that patient would receive screen positive result

33. Case 3
A 29 year old patient had an NT of 4.4mm at 12+5 weeks gestation
You offered NIPT and she accepted
NIPT results were normal. She is now 14+2 weeks gestation
What are the next steps?

34. Genetic counselling is recommended Patient likely to be offered:
Chromosomal microarray
Genetic testing for other single gene conditions
Level II ultrasound
Fetal echocardiogram

35. Non-Invasive Prenatal Testing Pearls
Consider offering NIPT to high risk women
Consider NIPT as a screen of higher sensitivity than current screening if your patient is willing to pay for the test
Not a diagnostic test
Not an all purpose genetic test, only gives info on specific chromosomes, and so not indicated in all circumstances

36. Prenatal Screening Summary
Offer all pregnant women, regardless of age:
PN screening for fetal aneuploidy (trisomy 13, 18, 21) through FTS, IPS, SIPS or Quad screening
Second trimester ultrasound for dating, assessment of fetal anatomy and detection of multiples
Maternal age should not be used as the basis for recommending invasive testing when non-invasive PN screening is available
Prenatal testing menu continues to evolve and expand with new screening and diagnostic tests
More, complex options may add to patient’s decisional conflict

37. Don’t forget
Take a family history to identify familial and/or ethnicity-specific disorders and screen accordingly
Consider consanguinity and screen and test accordingly
Refer or consult genetics when in doubt

38. Resources NIPT GECKO on the run GEC-KO website: Genetics Education
Society of Obstetricians and Gynaecologists of Canada (SOGC) national clinical guidelines in prenatal genetics visit and scroll down to Genetics.
Recent Reviews:
Cuckle H, Benn P, Pergament E. Cell-free DNA screening for fetal aneuploidy as a clinical service Clin Biochem. 2015; [Epub ahead of print]
Bianchi D and Wilkins-Haug L. Integration of non-invasive DNA testing for aneuploidy into prenatal care: What has happened since the rubber met the road? Clin Chem 2014; 60:1