Maternal screening for aneuploidy disorders using maternal serum hormonal immunossay levels & statistical risk algorithm is recommended to be used as a universal process as per ACOG & SOGC. Maternal blood has circulating fetal DNA which can be targeted in screening molecular tests like Non-Invasive prenatal testing(NIPT) for identifying aneuploidy. However, confirmatory tests still are cytogenetics (karyotyping) based tests using sample from amniocentesis or CVS.
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Maternal screening for fetal Aneuploidy- Update on Laboratory Tests
1. Maternal Screening –Maternal Screening –
Update on Laboratory testsUpdate on Laboratory tests
DR RAJESH V BENDRE
Chief Pathologist,
MD(path), DNB(path), DPB
2. GENETIC Abnormalities
3-5% of babies have a major birth defect or mental retardation
Congenital anomalies account for 8–15% of perinatal deaths and 13–16% of
neonatal deaths in India.
Risk in Indian population for Trisomy 21 Down’s syndrome is 1: 853
Trisomy 18 Edward syndrome Risk 1: 8,000
Trisomy 13 Patau syndrome Risk 1 : 5,000
Congenital Defects
Second most common major congenital defect neural tube defects
1-2 : 1000
- Congenital malformations at birth in Central India: Amar Taksande et al. Indian J Hum Genet. 2010
- Krishnamurty PS et al. Down's syndrome in Hyderabad, India . Acta anthropogenet 1985;9(4):256-60.
Rationale of prenatal screening
3. Who should be screened ?
Conditions causing increased risk for chromosomal anomalies
Maternal age ≥ 35 yr, ≥ 31 yr with twins
Previous Bad history
Autosomal trisomy,
Repetitive first trimester abortion
Major structural defect
Isolated structural anomalies
Familial genetic disease
History of parental consanguinity
Maternal - Diabetes Type I, Viral infections
Rationale of prenatal screening
Ideally, all women should be offered aneuploidy screening before
20 weeks of gestation, regardless of Maternal Age.(ACOG 2007)
5. Non- Invasive Prenatal Screening –
Use of Hormone Measurements -
•Hormonal changes contribute to the
physiological maternal adaptations during
gestation.
•These hormonal changes are different in pathological
pregnancies and may be monitored for diagnosis or risk
prediction of gestational diseases, taking into account
both the hormonal levels and the preexisting maternal risk
factors.
• Evidence concerning the applicability of
hormone measurements to the risk assessment,
early diagnosis, and management of
pregnancies complicated by Down’s syndrome,
fetal growth restriction (FGR), preeclampsia,
preterm delivery, and diabetes mellitus.
Rationale of Prenatal screening
• Free beta HCG and HCG from placenta
• PAPP-A from placenta
• AFP from fetal yolk sac and liver
• UE3 from fetal adrenal
• Inhibin A – placenta & gonads of mother
6. Pre-Natal Diagnostic Tests
Aneuploidy Screening Test – Challenges
- should be highly specific with low false
positive rate
- Sensitivity of the test is ascertained by
detection rate
- As the detection rate increases, the false
positive rate also increases
- Statistical risk calculation
- Use of Population specific MOM
- Use of established “cut off levels” for
assessing detection & false positive
rates for the disease.
7. Screening test – Risk Calculation
What is MOM & Why ??
• To equalize and determine a “reference” range during pregnancy.
When there are differences in hormone levels – Day by Day – Week
by Week
• For maintaining similarity of reporting in different labs & also
compensates for analytical variation in hormone
•Population specific Medians as per patient & Gestational Age & for
each hormone.
•These medians are fed in software & periodically updated &
monitored
MoM’s need Adjustment / Correction for:
– Patient’s weight, Maternal age,
Smoking, Diabetes, Race , Twin
pregnancies, IVF/ART
Screening test – Risk calculation MOM Correction
8. Hecht CA and Hook EB. 1996. Rates of Down syndrome at live birth by one-year maternal age intervals a proposed revised rate schedule
for use in genetic and prenatal screening. Am J Med Genet 62:376-385.
AGE wise Maternal Risk– Chromosomal Anomalies- Step 1
Screening test – Risk calculation- 3 steps Approach
Screening test – Risk
calculation- 2 & 3 steps
9. Recommended Cut-off levels for Software–
For Trisomy 21– 1 : 250 (190 to 400)
For Trisomy 18– 1 : 100 (100 to 300)
For open NTD–AFP MOM more than 2.5
Screening Test – Risk
calculation
First And Second Trimester Evaluation of Risks trial (FASTER-2005)- Prospective USA study looking at
performance of various combinations of screening markers in the first and second trimesters
10. PRENATAL SCREENING
Clinical Information Needed
Date of Birth: for age related risk
Gestation: by LMP or by ultrasound recommended
Maternal Weight: marker levels decrease with increasing maternal weight,
most important for AFP
Diabetic Status: maternal IDDM increases risk of ONTD while MSAFP
tends towards lower levels
Race: Blacks have higher MSAFP and lower population prevalence of
ONTD
USG Findings: for NT, CRL, Multiple Gestation- AFP increases with
number of babies; serum screening for DS not available for multiples
11. Laboratory QC Criteria
Alpha-Fetoprotein [AFP] Assay
• The coefficient of variation should not exceed 5%. The
accuracy should be within 3% from lot to lot.
Chorionic Gonadotropin [hCG] Assay
• The coefficient of variation should not exceed 5%. The
accuracy should be within 3% from lot to lot.
Unconjugated Estriol [uE3], PAPP-A, Free B-HCG Assay
• The coefficient of variation should not exceed 7%. The
accuracy should be within 5% from lot to lot.
-Monthly monitoring of individual parameter MOM
- Monthly monitoring of positivity rates for T21, T13/18,
NTD
- Quarterly monitoring of individual parameter MOM in
comparison with shared database, there by updating the
MOM as per Indian population
15. PRENATAL DIAGNOSIS OF FUTURE
Non-Invasive Fetal Trisomy Test
Method– Next Generation Sequencing
Trisomy 21 (Down's syndrome), 18 (Edwards syndrome), and 13 (Patau
syndrome)
Accuracy of over 99%
Other applications-Monogenic Disorders
Beta Thalassemia
Hemophilia
Limitations-
Test failure - low fraction of fetal DNA in the sample (<4%), the non-
invasive prenatal test cannot be reported. Low fetal fraction – occurring in
0.5–7% of cases is more likely at early gestations (8- 10 weeks) and in
obese patients.
Placental Mosaicism- There is good evidence that the source of the
cffDNA is the placenta. It is known from CVS data that abnormal cell lines
can be present in the placenta that are not present in the fetus (in
approximately 1% of CVS samples),
16. PlGF
Pre-eclampsia screening
(Alpha Software)
Detection Rate False positive rate
Quadruple markers plus second trimester PlGF 45% (all pre-eclampsia),
52% (early pre-eclampsia)
5%
First trimester PAPP-A and Mean Arterial Pressure
MAP together with the quadruple test markers plus
a measurement of second trimester PlGF
51% (all pre-eclampsisa) and 64%
(early pre-eclampsia)
5%
Placental Growth Factor (PlGF) is produced by the placenta. PLGF is an angiogenic factor, which is a
vasodilator that increases the diameter of existing arteries. Low levels of circulating free angiogenic factors
contribute to vascular endothelial dysfunction, which is one of the symptoms of pre-eclampsia.