This document discusses prenatal diagnosis techniques including screening tests and diagnostic tests. Screening tests include ultrasonography, maternal serum screening which tests biomarkers, and alpha-fetoprotein. Diagnostic tests include invasive techniques like amniocentesis and chorionic villus sampling, as well as non-invasive techniques using cell-free fetal DNA in the mother's bloodstream which can be used to detect genetic disorders as early as 6 weeks into the pregnancy. Cell-free fetal DNA is isolated from maternal plasma through size fractionation methods and can be used for applications like fetal sex determination and rhesus blood grouping.

1. PRENATAL DIAGNOSIS
PRESENTED BY :
THARAA MOHAMMAD RAND SLEIMAN
RUBA MAHMOUD QUSSAI ABBAS
SUPERVISED BY: DR . RENWAH AL-SAYYED

2. Prenatal diagnosis
When ? Why? And how ?

3. What’s the difference ??

4. SCREENING TESTS

5. SCREENING TESTS

6. ULTRASONOGRAPHY (USG)
Started at 6 WEEKS 16-20WEEKS looking for anomalies
NuchalTranslucency (NT) (2.5ml)

7. BLOOD TEST
 maternal serum human chorionic
gonadotropin [βhCG],
 pregnancy-associated plasma
protein [PAPP-A]
 Human placental lactogen [HPL]
 Rh factor
 HIV
 HBV
 Rubella
 Syphlis
Biomarkers and blood test

8. MATERNAL SERUM HUMAN CHORIONIC
GONADOTROPIN [ΒHCG]
Quantitative?? Qualitative??
immunometric assay

9. PREGNANCY-ASSOCIATED PLASMA PROTEIN (PAPP-A)
PAPP-A concentrations early in pregnancy have
been associated with:
 Down syndrome
a high rate of fetal loss
poor fetal growth
 premature delivery

10. SCREENING TESTS

11. ALPHA FETOPROTEIN (AFP)
 Yolk sac in first trimester
 Fetal liver in second and third trimester
 MSAFP normally increase from 12-32 weeks and it is most sensitive between 16-18 weeks

12. Maternal Serum Estriol(uE3)
 urinary and serum estriol
 in trisomies and in neural tube defects associated with adrenal hypoplasia

14. Dimeric inhibin-A
 negative feedback regulator of FSH secretion
 Inhibin is secreted by the placenta and corpus luteum

15. SCREENING TESTS

16. DIAGNOSTIC TESTS

18. 1- Amniocentesis
Invasive tests
Mechanism
Time
Risks
Investigations
Hydroamnios

19. 2-Chorionic Villus Sampling
Invasive tests
Mechanism
Time
Risks
Investigations

20. 3-Umbilical Blood sampling :
Invasive tests
Mechanism
Time
Risks
Investigations
Rh incompatibility

21. 4-Fetoscopy
Invasive tests
Mechanism
Time
Risks
Investigations
Hydronephrosis
FFTS

22. Technique Timing
Miscarriage
risk
Applications
Amniocentesis 15–17 weeks 0.5 %– 1 %
 chromosome analysis
 diagnosis of open neural tube
defect
 molecular genetic diagnosis
 biochemical diagnosis
Chorionic villus
sampling
11–14 weeks ~1%
 chromosome analysis (karyotyping)
 molecular genetic diagnosis
 biochemical diagnosis
Umbilical Blood
sampling
From 16–20 weeks ~1 %
 chromosome analysis
 hematological and biochemical
diagnosis

23. Non-invasive prenatal diagnosis (NIPD):
1. Ultrasonography:
 Diagnostic USG and screening USG
 Types of Ultrasonography:
 doppler
 Colour doppler
 Three dimential ultrasonography

24.  Diagnostic USG assess fetal proportions:
sex, position, growth; placenta, amniotic fluid
 Conditions detected by ultrasound:
 Neural tube defects
 Dawn syndrome
 Body wall defects
 Growth disturbances
 Major limb abnormalities
 Oligo- or polyhydramnios
 Major organ abnormalities
Non-invasive prenatal diagnosis (NIPD):
1. Ultrasonography:

25. Non-invasive prenatal diagnosis (NIPD):
 prior to in vitro fertilization at the third day
of fertilization DNA is extracted and
tested The embryo without genetic
disorder are implanted into mother.
 Karyotyping
2. PGD Preimplantation Genetic Diagnosis

26. Non-invasive prenatal diagnosis (NIPD):
3.Fetal Cells in Maternal Circulation
 Types fetal cells in maternal circulation:
(Placental cells,White blood cells and immature red blood cells with nuclei)
 Enter the bloodstream (6 and 12 weeks) and it can be detected in maternal
plasma from the 6th week of gestation.
 Useful for detection of genetic disease.
 Fetal cells are only 1/100,000 in mother’s blood

27. Non-invasive prenatal diagnosis (NIPD):
4.Cff DNA/RNA in Maternal Circulation
• Circulating cell-free DNA 
apoptotic cells, necrotic cells
• 10%
• Placental microparticles
• 28th
• 143bp
• mRNA-Y chromosome

28. SECOND: verify the presence of fetal material in the
maternal circulation (detection of paternally inherited
sequences e.g., sequences located on the Y
chromosome, paternally inherited single-nucleotide
polymorphisms (SNPs), epigenetic markers)
FIRST: cffDNA and cffRNA have to be
isolated and purified from maternal plasma

29. SAMPLE COLLECTION
AND PROCESSING
• EDTA
• Formaldehyde to prevent
lysis of maternal cells
• Centrifugation
EXTRACTION OF CELL
FREE FETAL DNA / RNA
• Size fractionation

30. Plasma DNA isolation is
performed manually with
commercially available kits
EpiQuik™ Circulating Cell-Free DNA Isolation Kit
Quick-cfDNA™ Serum & Plasma Kit
NextPrep-Mag™ cfDNA Isolation Kit

31. The EpiQuik™ Circulating Cell-Free DNA Isolation Kit utilizes magnetic beads
based size-fractionation technology to isolate circulating cell-free DNA
(ccfDNA) from mono-nucleosomal and di-nucleosomal complexes in
plasma/serum samples.The isolated ccfDNA can be directly used for real
time-PCR and DNA library preparation suitable for next generation
sequencing.The kit has the following advantages:
 Uses innovative magnetic bead based size-fractionation technology for
selective isolation of circulating cell-free DNA from plasma/serum that is
mainly 170 bps in size.The isolated DNA can be directly used for both
qPCR and NGS DNA library preparation.
 Fast and straightforward procedure can be finished within 2 hours. No
gels, columns or centrifugation is needed.
 Efficient removal of proteins, salts, nucleases, PCR inhibiting substances,
and other impurities such as polysaccharides, polyphenols and lipids.
 Sensitive and efficient DNA capture enables successful isolation with high
recovery (>80% of input monoucleosomal DNA), even when the
quantities of starting material are limited (as low as 0.1 ml).
 Manual and automation friendly – Scalable for single tube or 96-well plate
formats.

32. CLINICAL APPLICATIONS
 Fetal sex determination: identification of Y chromosome sequences in
maternal plasma  7th week
 Rhesus blood grouping: RT-qPCR  reliable detection of RHD
sequences using cffDNA.
 Single-gene diseases: applying PCR and mass spectrometry