Prenatal screening and diagnosis of fetal abnormalities like Down syndrome is important given the significant morbidity, mortality, and financial costs associated with these conditions. New screening techniques using cell-free fetal DNA analysis from maternal blood, known as non-invasive prenatal testing (NIPT), have been shown to detect Down syndrome and other trisomies with over 99% accuracy and a false positive rate below 1%. While NIPT has advantages over traditional screening by being non-invasive and having higher detection rates and lower false positives, diagnostic testing through amniocentesis or CVS is still recommended for positive NIPT results to confirm the diagnosis. NIPT is becoming the standard of care for high risk pregnancies but further validation is still needed

1. Prenatal Diagnosis of
fetal abnormalities
Dr Ashok Rattan, MD, MAMS
Chief Operating Officer &
Medical Director,
Star Metropolis Clinical Lab
Dubai, Sharjah, RAK

2. BOY or GIRL ? With Blue Eyes ?? Tall or short ??
Is all coded in our genes and NOT in our STARS

3. • Gene: A gene is a stretch of DNA whose
sequence determines the structure and function
of a specific functional molecule (usually a protein)
• Chromosome: Genes are located in the cell
nucleus on chromosomes
• Karyotype: ordered arrangement of
chromosomes in order of their size
– Human have 23 pairs of chromosomes [one set from each parent]
– 22 are autosomal, last pair sex chromosome; 46, xy or 46,xx
• Aneuploidy refers to cytogenetic abnormalities
in which entire or part of one or more
chromosome is added or deleted.
– Autosomal or sex chromosome aneuploidy

4. Prevalence of Aneuploidy

5. "Observations on the Ethnic Classification of Idiots"
"Mongolism"
John L H Down
1866
Jérôme Lejeune
1959

6. Three different patterns of
chromosomes can cause Down’s
syndrome

7. Down Syndrome
47,xx or xy, +21
• Commonest chromosomal abnormality
• Most frequent form of intellectual disability
• Characterized by:
– Severe learning disability (IQ about 40)
– Short stature
– Characteristic Facial features
– Heart defects ( 40 to 50 %)
• Caused by a demonstrable chromosomal
abnormality

8. Rationale for screening
• Frequent occurrence
– 1 in 634 live births
• More common with advanced maternal age
• Significant burden of increased morbidity &
mortality, besides financial stress
• Diagnostic tests readily available
• 40% of affected pregnancies are lost
spontaneously

9. Down Syndrome
Why terminate electively ?
• Associated with significant morbidity &
mortality
• High financial & psychological cost to family
• Severe learning disability (IQ < 40)
• Heart defects (40 to 50%)
• Intestinal malformation (10%)
• Vision & hearing problems (50%)
• Increased frequency of infection

10. Candidate for prenatal screening
• High maternal age
– Risk is non linear
– 1 in 1500 in young, 1 in 10
in 48 yrs old mother
– Risk is constant between
15 to 25 yrs
– 14% of pregnant mothers
were 35 yrs or older &
accounted for 51% of all
Down syndrome births;
oldest 5% for 30%
– Effect of paternal age is
uncertain

11. Importance of Screening All Pregnant
Women
Majority of
babies born with
Down syndrome
are in women
under 35 years
old
Majority of
babies born with
Down syndrome
are in women
under 35 years
old

12. 63, 398 new born babies in Dubai born between 1999-2003
24,250 UAE nationals; 39,148 non UAE nationals
141 karyotyping confirmed cases of Down Syndrome
139 trisomy 21; 1 translocation , 1 mosaic
Overall 1 in 449 live births (2.2 per 1,000)
In UAE nationals 1 in 319 (3.3 per 1,000)
In non UAE 1 in 602 (1.66 per 1,000)

13. Age distribution among UAE and non
UAE mothers with Down Syndrome child
Mean maternal age in UAE nationals 33.48 + 8.08 yrs
41.66 % of mothers were > 35 yrs of age

14. American College of Obstetric &
Gynaecology 2007
• All women be offered aneuploidy screening before 20
wks of gestation
• All women should have option to invasive testing
regardless of maternal age
• Fetal karyotype rather than serum screen should be
considered in women of any age at high risk
– A previous pregnancy complicated by fetal trisomy
– At least one major or two minor fetal abnormalities
in current pregnancy
– Chromosomal translocation, inversion or aneuploidy
in themselves or in partner

15. Screening Tests
• Maternal serum markers
– Serum marker pattern is related to poorly
functional fetal tissue with compensatory maternal
hyperfunction
• Low alpha fetoprotein
• Low unconjugated estriol
• High maternal serum human chorionic
gonadoropin
• High Inhibin A
• Ultrasound for nucheal translucency
• other risk factors (ethnicity, maternal age,
maternal weight, DM, Smoking)

16. PAPP-A levels increase by
30 to 50% per wk between
10 to 13 wks
Free or total hCG reach
Peak At 10 wks & decline
by 10 to 30 % /wk free
or 5 to 10% total
Pregnancy Associated Plasma Protein A
Blue line indicates unaffected pregnancy, Red line indicated Down Syndrome affected pregnancy

17. 1st
trimester Combined test
• Sonography:
– Nuchal translucency
– Gestational age: crown rump length
• Serum markers:
– Pregnancy associated plasma protein A
(PAPP-A)
– Free or total hCG hormone
• Time: 9 to 13 wks of gestation
• Diagnostic test for screen positive:
– Chorionic villus sampling

19. Integrated test
• A. Full integrated test
• B. Serum integrated test
– PAPP A
– hCG
– Alfa Fetoprotein
– Unconjugated estriol (uE3)
– Inhibin A

20. Increase by 15 to 20%/wk by 20 to 25%/wk
Decrease exponentially from peak nadir at 17 wks
At 15 wks

21. Quadruple Test
• Best screening option for women who
present late for prenatal care (15 to 18
wks)
• Four maternal blood biomarkers tested
– Alfa fetoprotein
– uE3
– hCG
– Inhibin A

22. Pregnancy Risk Screens
Estimate Risks Based Largely on
phenotypic factors
hCG
AFP
uE3
Inhibin A
PAPP-A
NT

23. Screen Positive
• When risk of Down syndrome in > 1 in 250
• Report provides actual calculated risk (>1 in
50)
• If ultrasound was not performed, do it now
• Rule out other causes : multiple pregnancies
• Double check TRF and information entered
• Repeating Serum screening tests is not
recommended

25. FPR=5.0% FPR<0.1%
Non Invasive
Prenatal Test
Evolution of Trisomy Assessment
ACOG Practice Bulletin No. 77. Obstet Gynecol 2007;109:217-27.
Detection rate for Down syndrome

26. Invasive Diagnostic Test for
Screen Positive
• Diagnostic test must be offered
– Karyotyping on sample obtained following
either
– Amniocentesis or

27. Non Invasive Prenatal Test
NIPT

28. Sources of Fetal DNA in
Maternal Blood
 Cells (~1 fetal cell per 1 billion total cells)
• Persist between pregnancies
 Cell-free DNA (Not fetal per se)
• 150-200 base pairs in length
• By 10 weeks’ gestation
~90% of total is maternal
oprimarily from apoptosis of blood cells
~10% is from the pregnancy
oprimarily from apoptosis of placental cells
• Half life < 20 minutes
• Undetectable < 2 hours postpartum
• Has been used for years for fetal RhD genotyping

29. Goals
of NIPT
Reduced
risk to
fetus
Reduced
anxiety
Increased
detection
Reduced
false
positives
(Available
to all
women)
Verinata

30. Performance Comparison
CVS Amnio Sequential NIPT
Timing 11-13 weeks ≥16 weeks 10-22 weeks ≥10 weeks
Procedure Invasive Invasive Ultrasound and
maternal blood
Maternal
blood
Risk 1%
miscarriage
0.1%
miscarriage
None
to pregnancy
None
to pregnancy
Sensitivity >99%
for
aneuploidy
>99%
for
aneuploidy
90%
for Down
syndrome
>98%
for Down
syndrome
Specificity >98%
for aneuploidy
(FPR <2%)
>99%
for aneuploidy
(FPR <1%)
95%
for Down
syndrome
(FPR 5%)
>99%
For Down
syndrome
(FPR <0.5%)
Turn around <2 weeks <2 weeks <1 week <2 weeks

31. What is NIPT
• 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)

32. NIPT
• NIPT measures circulating cell free fetal DNA
(ccffDNA) present in maternal blood (from
trophoblast)
• Comprises ~10% of DNA in maternal blood
• Increases with gestational age
• ccffDNA analysis determines if normal, higher, or
lower than expected quantity of particular DNA
sequences found on select chromosomes (13, 18, 21,
X, Y)
• Performed on maternal blood sample
• As early as 10 weeks’ gestation

33. What’s the evidence for NIPT?
• 7 studies of “high risk” women
• High risk:
– Screen positive
– AMA (≥35 yrs)
– Ultrasound findings
– Family history indicating increased risk
• Previous pregnancy with aneuploidy

34. What’s the evidence for NIPT?
By far most accurate performance for Trisomy 21 / 18
Benn et al, Ultras Obstet Gynecol 2013, 42: 15-33

35. What’s the evidence for NIPT?
• 4 studies on unselected pregnancies
– Most mixed risk, some after pos screen,
AMA, fewer with neg or no screen
– > 14,000 pregnancies total, largest (11,000
significant loss to follow up)
– Similar performance
• Not yet validated in low risk women,
triplets or higher, pregnancies
conceived with egg donation
Benn et al, Ultras Obstet Gynecol 2013, 42: 15-33

36. What’s the evidence for NIPT?
• Failed results
– 6.1% (0.8-9.9) untested for insufficient sample
quality
– 2% (436/22,222) no result after testing
– Rarely happens with conventional screening
– ccffDNA decreases with increased maternal BMI

37. Summary of evidence for NIPT
Disorder Sensitivity FPR
Down syndrome
Trisomy 21
99-100% ~0.1%
Trisomy 18 97-100% ~0.1%
Trisomy 13 79-92% ~0.1%
Sex chromosome
differences
94-99%

38. What do the experts say?
• American College of Obstetricians and
Gynecologists 2012
– Has been validated in industry sponsored
studies on at risk populations
• History of prior pregnancy with trisomy
• Positive multiple marker test
• Parental balanced Robertsonian translocation
with increased risk of fetal trisomy 13/21
• Maternal age >35
• Fetal ultrasound findings with increased risk of
aneuploidy
ACOG Committee Opinion: Dec 2012

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

40. 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

41. NIPT
What is done ?
• 2 x 10 mL of blood is collected in
special “Streck” tubes [ mix well]
• After 10 weeks of pregnancy
• Results after 10 working days
• Report on
– Trisomy 21, 18 and 13
– Sex aneuploidy : XO, XXY, XXX
– Sex of baby

42. Key differencesKey differences
Different approaches to cfDNA analysis
Binary +/- result
based on z-score
Risk classification
and risk score
Massively Parallel
Shotgun Sequencing
(MPSS)
Directed Approach
NIPT Technology
42

43. Branded market names for the
NIPT tests
• Harmony (LabCorp)
• MaterniT21+ (Sequenom)
• Verifi (Verinata)
• NIFTY (BGI)
• Praena Test (Lifecodexx)
• Panorama (Natera)

45. Testing Beyond Common
Trisomies
Patient wants
current
standard of
care
Clinical Test
to Consider
NIPT
(T21, T18, T13)
Patient wants
comprehensive
genetic
evaluation
Wants full genetic
evaluation of fetus
Invasive testing
with microarray
Only concern is T21,
T18, T13, and sex
aneuploidy
(after counseling re: sex
chromosome aneuploidy)
Wants definitive
information
Invasive testing
with karyotype
Wants safe test
NIPT
with XY
45