Down syndrome (trisomy 21), which has an incidence of 1 in 800 live births, is considered to be the most frequent etiology of mental retardation and it is the predominant reason for women seeking prenatal diagnosis. Trisomy 21 is used as a benchmark because it is the most common aneuploidy compatible with life and is associated with mental retardation and serious congenital anomalies. Currently used screening tests for aneuploidy are based on the assessment of fetal sonographic markers and/or the evaluation of biochemical markers in the maternal circulation during the first and second trimester. Screening test based on the combination of nuchal translucency assessment and biochemical markers at 11+0-13+6 weeks of gestation may detect 90-94% of pregnancies affected by Down syndrome at a false positive rate of 5%. The current gold standard for diagnosis of trisomy 21 is provided by invasive sampling of fetal genetic material through chorionic villus sampling (CVS) or amniocentesis followed by conventional cytogenetic or DNA analysis; however, both procedures are associated with an increased risk of fetal loss of about 1% and therefore they are recommended for pregnancies considered to be at high risk of fetal trisomy 21. To minimize the risk of fetal loss and false positive cases, Tri-Gene test is designed for the early prenatal detection of fetal trisomies of 21, 18, 13 and cases of other chromosomal aneuploidies. The non-invasive test carries no risk of causing miscarriage and intrauterine infection, and is highly sensitive with accuracy over 99%.

2. Dr. Sanjida Ahmed
Director, Research

3. Overview

4. Prenatal Diagnosis
Method Invasive/Non- Pregnancy (Weeks) Risk & Detection rate
invasive
First-trimester serum Non-invasive 11-13 65%-70% detection rate
screening
Second-trimester serum Non-invasive 15-19 69%-81% detection rate
screening
Fetal nuchal translucency Non-invasive 11-13 64%-70% detection rate
(NT)
Serum integrated screen Non-invasive 11-13 & 15-19 85%-88% detection rate
Serum integrated screen Non-invasive 11-13 & 15-19 94%-96% detection rate
+ NT
Amniocentesis Invasive 16-21 0.5-1% miscarriage
Permission required
Chorionic villi aspiration Invasive 10-13 1-2% miscarriage
sampling
Permission required
(CVS)
Percutaneous umbilical Invasive 18-24 1-2% miscarriage
blood
Permission required
sampling (PUBS)
Fergal D, Jacob A, et al. The New England Journal of Medicine, 2005

5. P otential L imitation of Current
High false positives T False Negative- BIG PROBLEM
esting
Screening tests may have up Screening can detect 94- 96%
to a 5% false positive rate leaving other 4-6% which
potentially creating anxiety could be positive and lose
for many women every year. detection credibility.
Safety concerns
Safety concerns
Many women
Many women
decline invasive
decline invasive
procedures (CVS,
procedures (CVS,
amnio) because of
amnio) because of
risk concerns.
risk concerns.

6. Research on Non-invasive Prenatal
Diagnosis (NIPD)

7. Cell-free Fetal (cff) DNA
 Cell free DNA (cfDNA) are short DNA fragments
 In pregnancy, cfDNA from both the mom and fetus are in maternal blood
 Amount of fetal cfDNA present is a small fraction of the maternal cfDNA

8. Origin of Cell-free Fetal (cff) DNA
•Hematopoietic cells
•Placenta
•Direct transfer of DNA molecules

9. Evolution of NIP T
D echnology

10. Revolutionary DNA Technology
 Random sequencing (also called massively parallel shotgun sequencing)/
Next Generation Sequencing
 Directed DNA analysis/ Digital Analysis of Selected Regions (DANSR-)
NGS
NGS
Similar to Sanger’s sequencing
Similar to Sanger’s sequencing
Generates far more sequence
Generates far more sequence
 Recent advancement enables rapid sequencing of a large
stretches of advancement enables rapid sequencing of a large
 Recent
DNA
stretches of DNA
 More sensitive to small changes in genomic representation
 More sensitive to small changes in genomic representation

11. Principle of Next Generation
Sequencing
1. Incorporation
1. Incorporation
1. incorporation 2. Elute and scan
2. Elute and scan
3. Cleavage
T T G
G
C C X X
X X
X
C C A
A
X X X
X

12. Principle of Analysis
Plasma cf-DNA
extraction
Bioinformatics
Sequencing &
alignment

13. Principle of Analysis
Chromosomes
counting
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Fragments for Chromosome N
Coverage
computation
% Chromosome N =
Total Fragments

14. Statistical Analysis
100 cell‐equivalent free DNA / ml plasma: 95 from mother, 5 from fetus.
Pregnant woman with healthy fetus Pregnant woman with trisomy 21 fetus
Maternal Chr 21: Fetal Chr 21: Maternal Chr 21: Fetal Chr 21:
95X2=190 5X2=10 95X2=190 5X3=15
In maternal blood
In maternal blood In maternal blood
In maternal blood
Total Chr 190+10= 200
Total Chr 190+10= 200 Total Chr 190+15= 205
Total Chr 190+15= 205
Statistics for t score and L score

15. Clinical Validation Process
Pregnant women with high
risks of T21, T18, T13
Blood sampling and coding
Amniocentesis or CVS
Double blind test
Third part lab
NIPD for fetal T21, T18, T13
Karyotype analysis
Result analysis
Statistics for sensitivity and
specificity

16. Clinical Validation Result
Non-invasive pre Fetal Karyotyping Analysis Total Samples
natal screening
Positive Samples Negative Samples
High risk 188 1 189
Low risk 0 3275 3275
Total 188 3276 3464

17. Clinical Validation Result
Non-invasive pre Fetal Karyotyping Analysis Total Samples
natal screening
Positive Samples Negative Samples
High risk 63 1 64
Low risk 0 3400 3400
Total 63 3401 3464

18. Clinical Validation Result
Non-invasive pre Fetal Karyotyping Analysis Total Samples
natal screening
Positive Samples Negative Samples
High risk 10 0 10
Low risk 0 3454 3454
Total 10 3454 3464

19. Advantage of the technology

20. Application of NIPD

21. Sample Needed
Sample Type Quantity Requirements Conservation and
Shipment
Plasma Separated from 5ml Stored in 1.5ml Storage at -80 ; Shipped in dry ice
maternal blood Eppendorf tube, and
sealed with 1cm wide
parafilm.
DNA Extracted from 5ml Storage at -80 ; Shipped in dry ice
maternal blood or blue ice
Maternal Blood 10ml in Streck Cell- Gently invert the tube Stored and shipped between
Free DNA BCT tube ten times immediately 18~25 within 3 days. Keep the
after blood sampling. tubes erect during shipping.

22. References
http://genomemedicine.com
www.intechopnen.com
www.bmj.com
www.AJOG.org
www.karger.com/fdt
http://humupd.oxfordjournals.org

23. Thank You