1. Non-Invasive Prenatal Testing
Developed by Dr. June Carroll, Ms. Shawna Morrison and Dr. Judith Allanson
Special thanks to the Children’s Hospital of Eastern Ontario’s Prenatal Genetics Team
Last updated April 2014

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 prenatal screening
Discuss and address patient concerns regarding prenatal screening
Facilitate patient informed decision making regarding prenatal screening options
Find high quality genomics educational resources appropriate for primary care

4. 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?

5. 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?

6. Case 3
A 36 year old G1P0 woman is very anxious about Down syndrome in this pregnancy because she had a second cousin with Down syndrome who passed away as a child.
Is NIPT a good option?

7. Case 4
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?

8. Prenatal Screening tests for: DS, T18, T13 and ONTD
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)

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. Non-Invasive Prenatal Testing (NIPT)
NIPT measures circulating cell-free DNA (cfDNA) from placenta present in maternal blood
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)
Dating U/S – 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 good is Non-Invasive Prenatal Testing?
Moving target
Currently literature is primarily from companies or those holding patents
Overall ranges
T21
T18
T13
Specificity (%)
99-100
Sensitivity (%)
98-100
97-100
79-100
Positive Predictive Value [PPV] (%)
90-95*
84*
52*
Negative Predictive Value [NPV] (%)
99.9 99
100
These numbers are in evolution
Range per company - all better than current screening
Pick up majority of cases but likelihood of true positive depends on frequency of condition.
CONFIRM the RESULT!!!!!!!
Sensitivity (also called the true positive rate,) measures the proportion of actual positives which are correctly identified as such (e.g. the percentage of sick people who are correctly identified as having the condition). Specificity measures the proportion of negatives which are correctly identified as such. (e.g. the percentage of healthy people who are correctly identified as not having the condition, sometimes called the true negative rate).
the positive predictive value, or precision rate is the proportion of positive test results that are true positives
NPV: the proportion of negative test results that are true negatives
For IPS: the PPV is probably around 10% for T21; the detection rate approaches 90% for T21
*ASHG Oct 2013 platform presentation – data from BGI China; 63,543 pregnancies

12. What is the evidence for Non-Invasive Prenatal Testing (NIPT)?
Studies emerging in low-risk population
Performance of NIPT in a general obstetrical population
False Positive Rate (FPR) for T21 and T18 combined was 0.5% vs 4.2% for standard screening
Positive Predictive Value (PPV) was significantly higher than that for standard screening, for both trisomy 21 (45.5% vs. 4.2%) and trisomy 18 (40.0% vs. 8.3%)
PPV - the percentage of patients with a positive test who actually have the disease
NPV – the percentage of patients with a negative test who do not have the disease
Specificity - ability of a test to correctly classify an individual as disease- free
Sensitivity - Sensitivity is the ability of a test to correctly classify an individual as ′diseased
Bianchi et al, NEJM 2014; 370:9

13. What is the evidence for Non-Invasive Prenatal Testing (NIPT)?
7 studies of “high risk” women
High risk:
Screen (IPS, FTS) positive
AMA (≥35 yrs)
Ultrasound findings suggestive of aneuploidy
Previous pregnancy with aneuploidy

14. What is the evidence for Non-Invasive Prenatal Testing (NIPT)?
By far most accurate performance for T21/18
Benn et al, Ultras Obstet Gynecol 2013, 42: 15-33

15. What do the experts say?
Society of Obstetricians & Gynecologists of Canada 2013
Non-invasive prenatal testing using massive parallel sequencing of cell-free DNA to test for trisomies 21, 18, and 13 should be an option available to women at increased risk in lieu of amniocentesis. Pre-test counselling of these women should include a discussion of the limitations of non-invasive prenatal testing.
Genetics Ctte Technical Update JOGC: Feb 2013

16. What do the experts say?
Society of Obstetricians & Gynecologists of Canada 2013
No irrevocable obstetrical decision should be made in pregnancies with a positive non-invasive prenatal testing result without confirmatory invasive diagnostic testing.
Genetics Ctte Technical Update JOGC: Feb 2013

17. Recommendations Offer all women:
Prenatal screening using either FTS, IPS or MSS (SIPS or Quad)
Fetal morphology scan at weeks’ gestation

18. Consider discussing Non-Invasive Prenatal Testing (NIPT) as an option 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 fetal nuchal translucency (NT) measurement of 3.5mm or greater 
Have had a previous pregnancy or child with aneuploidy
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].*
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.

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

20. Non-Invasive Prenatal Testing (NIPT) results
If the result is negative, this is reassuring
Your patient should still be offered:
MS-AFP between 15 and 20+6/7 weeks to screen for open neural tube defect, as NIPT does not screen for this physical anomaly
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, other soft markers, congenital anomalies)

21. 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

22. 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

23. 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% untested for insufficient sample quality
2% no result after testing (rarely happens with conventional screening)
cfDNA decreases with increased maternal BMI, twin pregnancies and where trisomy 18 and 13 are present
Limited data on performance in IVF pregnancies

24. Limitations of Non-Invasive Prenatal Testing: Confined placental mosaicism
False positives (and negatives) are possible
Confined placental mosaicism
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. Non-Invasive Prenatal Testing (NIPT) Landscape
Increasing demand from women
Increasing uptake in most (urban) centres
3 separate companies, 3 separate technologies
Has NIPT become “standard of care”?
Costs between $795 and $1200
8-10 days for result

26. 11 to 14 week scan has value to pregnancy care
Where does Non-Invasive Prenatal Testing (NIPT) fit with respect to the 11 to 14 wk scan?
11 to 14 week scan has value to pregnancy care
Raised NT may suggest other chromosomal, genetic and structural disorders
Accurate dating/establishment of live fetus
Multiples/chorionicity affects management
Detects structural abnormalities

27. Non-Invasive Prenatal Testing (NIPT) and counselling
Pre- and post-test counselling is important
Consult genetics if unsure
Refer for genetic counselling when appropriate
Company websites:
Harmony Prenatal Test™ by Ariosa Diagnostics through Gamma-Dynacare
Panorama™ by Natera through Lifelabs
Verifi® Prenatal Test by Verinata

28. Ordering Non-Invasive Prenatal Testing (NIPT)
In Ontario, to order NIPT, first determine your patient’s funding eligibility by looking at the MOHLTC checklist, then:
Complete the checklist and pages 2 and 4 of the MOHLTC out-of-country testing prior approval form
Download and complete the requisition from the NIPT company of your and your patient’s choice
Selection may be based on the patient’s personal research and preference, your experience with a particular company, ease of access to a blood draw location
Send all three completed forms to the fax number or and be sure to indicate the patient is pregnant so approval can be prioritized accordingly
Once approval has been received, give the patient the approval letter and the NIPT requisition and she can then have her blood drawn

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
More rapid result from amniocentesis
What is her IPS risk?
1 in 2 versus 1 in 120
She is at 17 weeks gestational age
If NIPT is positive, guidelines recommend confirmatory diagnostic testing by amniocentesis – delays timing for diagnosis
In the event of an abnormal result, is termination of pregnancy an option for the couple?

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 36 year old G1P0 woman is very anxious about Down syndrome in this pregnancy because she had a second cousin with Down syndrome who passed away as a child.
Is NIPT a good option?

34. Case 3 She does not meet MOH criteria for coverage of NIPT
You can reassure her that having a second cousin with Down syndrome does not increase her risk to have an affected child
If she is very anxious, you can proceed with NIPT and she can pay out of pocket
If the patient herself had a previous affected pregnancy then she would be eligible for MOH funded NIPT 1a 1b 2a 2b 3b 3a 4a 4b
4a has Down syndrome
4b and 4a are second cousins – 5th degree relatives P

35. Case 4
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?

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

37. 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 a basis for recommending invasive testing when non-invasive PN screening is available
Non-Invasive Prenatal Testing (NIPT)
Consider offering NIPT to high risk women
Consider NIPT as a screen of higher sensitivity if your patient is willing to pay for the test
Best for Down syndrome (trisomy 21) (but can be wrong!)
so far, others not as good (T13 and sex chromosomes)
Effectively sampling the placenta (not fetal cells directly)
It is not a diagnostic test
Not an all purpose genetic test, only gives info on specific chromosomes, and so not indicated in all circumstances
NIPT is ok for T18, not as good as T21 but better than T13 and the sex chromosomes

38. 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

39. Useful Genetics Resources
GEC-KO website: Genetics Education
NIPT fact sheets:
Prenatal Screening Ontario Website
Society of Obstetricians and Gynaecologists of Canada (SOGC) national clinical guidelines in prenatal genetics visit and scroll down to Genetics.