The document discusses circulating cell-free fetal DNA (cffDNA) in maternal circulation, its applications in non-invasive prenatal testing (NIPT), and advancements in screening techniques since its initial discovery in 1997. It highlights the accuracy of NIPT for detecting fetal chromosomal abnormalities like trisomies with sensitivity and specificity nearing 100%, alongside challenges such as cost and limited availability. The document underscores the need for further studies, especially for average-risk pregnancies, and notes the potential for cffDNA testing to evolve into whole fetal genome sequencing.

1. NIPD
Ahmed El-Habashy
A.Lect.OB/GYN.Alex.Univ.

2. OUTLINES
• TERMINOLOGY
• TICHNIQUE &
SAMPLING
• APPLICATION
• ACCURACY
• RECOMMENDATIONS

3. What is cffDNA?
• 1st reporting of cffDNA
in the Maternal
Circulation  1997.
• From Placental
Apoptosis.
• cffDNA in Mother is 25
times > Fetus.
• Not contained within cells.
• Short life 3h.
Lo YM et al. Lancet 1997.

4. cffDNA  F/M FRACTIONS

5. NIPD = NIPT = NIPS
Diagnosis Testing Screening.
Although
• cffDNA discovered
Since 1997,
• FTS  Non-
Invasive Prenatal
Screening of Fetal
Trisomies.
• Its Implementation in
Practice was just
since 2011.
• NIPD = cffDNA
ONLY.

6. NIPD GENERATIONS
1st
Quantitative
MPS
SHOTGUN
TARGTED
2nd
SNP
K. Nicolaides Prenat Diagn 2013
Random MPSS
Selective Directed
Single Nucleotide Polymorphism

7. Massively Parallel Sequencing MPS
Shotgun MPSS Targeted / Directed MPS
• Millions of DNA
fragments From All
Chromosomes
• MostWidely Used
NIPT
• Digital ANalysis of
Selected Regions
DANSR
Sparks AB Prenat Diagn 2012
K.Nicolaides AJOG 2012
Ehrich et al Am J Obstset Gynecol 2011

8. MPSS VS DANSR
DANSR Use 1/10 cfDNA Fragments in MPSS
May ↑ Resampling
This ↑Throughput & ↓ Cost.
• ?!?!?WHETHER
DANSR or MPSS 
MORE ACCURATE
RESULS.
• Need Further Studies
to COMPARE BOTH
APPROACHES IN
the SAME COHORT
of PATIENTS.
RCOG 2014

9. STEPS of MPS
PCR
Amplification
SEQUENCING
ANALYTICAL
ALGORITHM

10. Chromosome Mapping
to a Reference Disomic Genome

11. SEQUENCING
Spatial Parallel Segregation
F/M cfDNA PROPORTIONAL RELEATIONSHIP

12. 2nd Generation NIPD
SNP
• Also a Targeted Sequencing.
• Not Need A Disomic Reference Chromosome.
Zimmermann Prenat Diag 2012
K.Nicolaides Prenat Diagn2013

13. SNP

14. cffDNA SAMPLING
• >10w GA ( SNP can at 9w).
• Spontaneous or Assisted Conception.
• 10-20 cc Maternal Venous Blood. (?! 2cc)*
• Results within 1-2 w.
• 5 different Companies (USA,Germany,China).
• PRICE : 800-3000 $.
*K.Nicolaides AJOG 2012

15. cffDNA APPLICATIONS
Not Present in the
Mother
Gender
RH
Paternity
(NIPP)
Single Gene
Disorder
In Relation to What is
Present in the Mother
Autosomal
Trisomy
Sex Chromosomal
Aneuploidy
Triploidy
Deletion

16. Autosomal Aneuploidy
Commonest
•T21
•T18
•T13
On Request in Some NIPT
•T16
•T22

17. Sex Chromosomal Aneuploidy
(Some NIPT)
• XO (Turner Syndrome)
• XXX (Triple X)
•XXY (Klienfelter Syndrome)
•XYY (Jacob Syndrome)

18. SO NIPD Can Detect ~ 85% of Fetal
Chromosomal Aneuploidy

19. Deletion Syndromes
Some NIPD
• 22q11.2 deletion syndrome (DiGeorge)
• Cri-du-chat syndrome (5p minus)
• Prader-Willi/Angelman syndrome (15q)
• 1p36 deletion syndrome

20. SensiGene
• RhD –ve Mother / RhD +ve Father.
• +ve Maternal Indirect Coomb (Sensitized).
• ± Hetrogenous Rh +ve Father.
• cffDNA♀RhD-ve 
= Mother
Can not Verify the presence of Fetal
DNA
RCOG 2014

21. SensiGene- SNP
• Fetal Identifier (FI) control is performed to
Compare M/F genotype.
• If ≥ 6 Markers are Observed  Verify True
RhD-ve ♀ Fetus:
o No Antenatal RhIG Prophylaxis .
o No Extra M/F Testing (± Invasive)
RCOG 2014

22. Fetal Gender
• If ♀ Fetus  50%
↓INVASIVE TESTING.
• If ♀ Fetus Early
Dexamethasone
↓Virilization
• If ♂ Fetus Early
cessation of
Dexamethasone.
Duchenne Muscle
Dystrophy
CAH
RCOG 2014

23. Single Gene Disorders
Paternally Inherited Allele
Autosomal Dominant
• Huntington Disease.
• Achondroplasia.
• Thanatophoric Dysplasia.
Autosomal Recessive
• ß Thalassemia.
• Cystic Fibrosis.
ABSENT Paternal
Allele  NO
INVASIVE TESTING.
RCOG 2014

24. NIPD Accuracy
• SPECIFICITY and SENSETIVITY CLOSE
TO 100% (>98%) FOR T21,18; Less for T13.
• FPR < 0.5%.
• 1-10% Chance of No Result (Sample Failure-
No Call Rate)  Mostly √ in Repeat Sample.
(Mostly FREE).
RCOG 2014

25. Possible Sources of ERRORS
FALSE-ve
• ↓ Fetal cffDNA
Fraction
oEarly GA:<10w 
<4-5%.
oMaternal Obesity.
FALSE+ve
• Confined Placental
Mosaicism (CPM):
Trisomic Placental
Cells BUT a
NORMAL FETUS.
Maternal Conditions:
o Chromosomal Aberrations.
o Malignancy.
RARELY  DISCORDANT
RESULTS
TWIN
Still Evolving
RCOG 2014

26. Maternal Weight VS cffDNA

27. cffDNA in Twining
MC
• Not only
Possible BUT
MORE
Effective.
DC
• CAN Calculate %
Fetal cfDNA
Fraction of EACH
FETUS.
If 1 Miscarries 
?!?
Its Fraction Change
Still Evolving
cffDNADx Zygocity *
*Qu JZ.et al. Clin Chem 2013
RCOG 2014

28. Comparison with Screening Tests
• Combined T21 FTS
= NT,ßHCG,PAPPA
 90% DR with 3%
FPR
• Sensitivity &
Specificity of cffDA
for T21≈100% .
↓INVASIVE
TESTING:
o COST
o LOSSES
K.Nicolaides Ultrasound Obstet Gynecol 2007

29. Prenatal Triosomy Detection Rate DR
FTS NIPD Invasive
(Karyotyping)

30. ACOG,SMFM,ACMG,ISPD
• cffDNA is some FORM
of CONTINGENT
Approach offering
testing to HIGH RISK
Pregnancies:
• Maternal Age ≥35Ys.
• Previous T21.
• Parental Balanced
Translocation.
• US Aneuploidy
Marker(s).
• +ve Biochemical
Screening test.
THE MOST EFFECTIVE
ANEUPLOIDY
SCREENING IN HIGH
RISK PREG.
ASOG,SMFM 2012
ACMG,ISPD 2013

31. SOGC
• >98% Detection Rate.
• <0.5 FPR.
• NOT A DIAGNOSTIC TEST.
• Can Not Replace US (NT).
• +ve NIPS  SHOULD do CONFIRMATORY
INVASIVE TEST BEFORE TOP.
• NIPT May REPLACE the CURRENT
MATERNAL SCREENING APPROACHES
IF:*
o ↓ COST.
o ↑ Studies in AVERAGE RISK PREG.
Sholud
Included in
Pre-Test Pt.
COUNSILING
*
*ACOG 2012
*SOGC 2013

32. NIPT IMPLEMENTATION
AGAINST
• ↑ COST : (COMMERCIALISATION and INTELLECTUAL
PROPERTY).
• FTS also Predict PET,FGR.
• FTS also Predict Many Non-Chromosomal Structural
Anomalies.
• If Contingent (done for +ve FTS)  MISS the FALSE –ve of
FTS.
• LIMITED STUDIES IN AVERAGE RISK* AND TWINS.
• ↓ INVASIVE TESTING  ↓ Fetal Medicine Trainee
SKILLS.
• Limited Geographical Lab. Distribution.
*K.Nicolaides AJOG 2012

33. NIPT IMPLEMENTATION
WITH
• NO F/M Harm.
• ↓ False –ve. (~ 38%*)
• ↓ False +ve of FTS 
• ↓ INVASIVE TESTING COST &
• ↓ INVASIVE TESTING Losses. (~ 66%*)
• IF REPLACE ßHCG,PAPPA  SAVE THERE
COST FOR NIPT.
• Multiplexing many patient samples in a SINGLE
SEQUENCING RUN ↓ Individual TEST COST.
*Garfield S J Manag Care Med. 2012

34. NIPD CAN ADJUST FTS RISK
Benn Calculator
• +ve NIPT  x 290 ↑ FTS Risk of T21
• -ve NIPT  x 110 ↓ FTS Risk of T21
Benn et al.,Ultrasound Obstet Gynecol ;2012

35. ACMG 2013
• No Doubt NIPS Costs WILL ↓.
• NIPS is Likely the 1st of major steps toward
the eventual application of WHOLE FETAL
GENOME SEQUENCING.

36. NIPT Market Competition
• Intellectual Property .
• Non-Profit Funding.
• ↓ Lab. Geographic
Distribution.
• Companies Litigation
• None of them FDA
Approved Yet ( in
Progress).
• No Insurance
Coverage.
• Stick-holders Should
Pay Attention to NIPD
Commercialization 
↑ Patient Access.
Ashwin A .Prenat Diagnos 2013
MARKET MONOPOLY
↑ COST

37. Trials
• Maternal Urine : cffDNA (MPS)*.
• Maternal Blood :
ocffRNA (more Fetus Specific)
oFetal Proteins.
oFetal CELLS.
Lo YM , K.Nicolaides Nat Med 2007

38. Take Home Message
• NIPD  High Accuracy + NO F/M Harm 
REVOLUTION in FETAL MEDICINE.
• NIPD MAJOR PROBLEM  ↑ COST 
FALLING  May REPLACE
BIOCHEMICAL MARKERS BUT CAN
NOT REPLACE US.
• NIPD are NOT DIAGNOSTIC  SHOULD
BE CONFERMED BY INVASIVE TEST.

39. THANK
YOU