This document discusses using cell-free fetal DNA in maternal blood to non-invasively screen for fetal chromosomal abnormalities like trisomy 21, 18, and 13. Current methods require invasive tests on fetal tissue that carry risks of miscarriage. The document outlines how cell-free fetal DNA can be detected in maternal plasma using PCR and sequencing. It notes the percentage of fetal DNA needed for accurate detection and limitations like confirmation with diagnostic testing.

1. FETAL DNA IN MATERNAL SERUM
DR KAPIL DEV

2. Trisomies
• Trisomy 21 is due to an extra chromosome 21 also called
Down Syndrome and is the most common trisomy at the time
of birth.
• Trisomy 18 is due to an extra chromosome 18. also called
Edwards Syndrome, is associated with a high rate of
miscarriage.
• Trisomy 13 is due to an extra chromosome 13. also called
Patau Syndrome, is associated with a high rate of miscarriage

4. How are trisomies diagnosed?
• Current methods of fetal genetic testing typically
involve obtaining samples of amniotic
fluid, placenta, fetal blood, or rarely, other fetal
tissues or fluids.
• The invasive techniques required for obtaining
fetal samples (eg, amniocentesis, chorionic villus
biopsy, fetal venipuncture, fetoscopy-guided
biopsy) place the fetus at risk of injury or death.

5. Chorionic villi sampling (CVS)
• A small amount of tissue from the developing
placenta.
• tested chromosomes.
• CVS is typically performed between 11 and 14 weeks
of pregnancy.
• CVS is associated with a small risk of miscarriage - 12%

6. Amniocentesis
• withdraws a small amount of fluid that
surrounds the fetus.
• tested for chromosomes.
• An amniocentesis is usually performed
around or after the 15 weeks of pregnancy.
• Amniocentesis is associated with a small risk
of miscarriage- 1%

7. • Complications associated with invasive prenatal
diagnostic tests:
•
•
•
•
•
•
Pregnancy loss (0.15%-1.0%)
PROM
Vaginal bleeding
Worsening sensitization of alloimmunization
Infection
Placental mosaicism

8. • Fetal DNA in maternal plasma or serum was first detected by PCR amplification
of a Y-specific fetal sequence, followed by the analysis with agarose-gel
electrophoresis and ethidium-bromide staining.
• In a subsequent study Lo et al
• Developed a real-time quantitative PCR assay to measure the concentration of
fetal DNA in maternal plasma and serum.
• This system is sensitive enough to detect the DNA equivalent of a single target
cell.
• Other advantages include a large dynamic range of over five orders of
magnitude, a high through put, and the use of a homogeneous amplification
and detection system that minimizes the risk of carry-over contamination.
• The main limitation of PCR analysis of Y-chromosomal sequences in maternal
plasma is that this approach can be used only in pregnancies involving male
fetuses.

9. Background:
Fetal Nucleic Acids in Maternal Plasma
•First report of free fetal DNA in maternal circulation. (Lo YMD et
al. Lancet 1997;350:485-7)
•Fetal DNA clears rapidly from maternal circulation after the
baby is delivered. (Lo YMD et al. Am J Hum Genet 1999;64:218-24)
•First report of free fetal RNA in maternal circulation. (Poon LLM
et al. Clin Chem 2000;46:1832-4)
•Prenatal diagnosis of fetal RHD status by molecular analysis of
maternal plasma. (Lo YMD et al. N Engl J Med 1998;339:1734-8)

14. LOW RISK PATIENTS
(HIGH RISK SHOULD BE SCHEDULED FOR
COUNSELING AT 10 WEEKS)
Blood draw for PAPP-A and
HCG at 9-12 weeks.
High Risk
> 1/65 risk for Down syndrome
Counseling- tests offered
·Invasive testing
·Maternal serum fetal
DNA test
·Complete Sequential
Ultrasound scheduled per usual
11 4/7 weeks to 14 weeks
(12 weeks optimal)
Intermediate Risk
1/66 to 1/2000 risk for Down
syndrome
Brief Counseling
·Sequential screen
> 1/270 risk -counseling
< 1/270 riskCounseling based on results
Low Risk
< 1/2000 risk for Down
syndrome
No further testing

18. Library preparation

19. Cluster Generation

20. Massively Parallel Sequencing

23. Data Analysis
• Alignment (chromosome matching) using human genome
database
• One matching error per 36 bases allowed
• Interpretation of results:
% of matches on chromosome 21
Z score for each sample

26. Trisomy Detection – Fetal fraction matters
 Trisomy detection via cell-free DNA analysis depends on the fetal fraction (proportion of
fetal to maternal cell-free DNA within the sample)
 The higher the fetal fraction the easier it becomes to detect aneuploidy
10% fetal DNA in circulation
Disomic Chr
Trisomy 21
Total: 100
Total: 105
(Maternal: 90)
(Fetal:
10)
(Maternal: 90)
(Fetal:
15)
20% fetal DNA in circulation
Disomic Chr
Trisomy 21
Total: 100
Total: 110
(Maternal: 80)
(Fetal:
20)
(Maternal: 80)
(Fetal:
30)

29. Some Limitations
• Diagnostic testing is recommended following
all positive results .
Chance pregnancy affected ≥ 50%
• Negative result does not ensure unaffected
pregnancy .
• Cost efficacy

31. THANK YOU