Dr Narendra Malhotra

1. SCREENING WHAT TESTS WHEN &
WHAT NEXT?
NARENDRA MALHOTRA
NEHARIKA M BORA
JAIDEEP MALHOTRA
KANIKA BANSAL
MANJEET MEHTA
Global rainbow health care India

2. Reference Material
• New WHO guidelines on antenatal care –
Systematic review BJOG 2016;123:519-28
• Guidelines by Government of western Australia
• SOGC guideline on Prenatal Screening
• RCOG / NICE Guidelines
• FOGSI expert group(TOG) 2017/2019

3. “I get by with a little help from my friends”
Dr S Suresh
Dr Ashok Khurana
Dr Pratima Radhakrishnan
Dr Deepika Deka
Dr Anita Kaul
Dr Feroza Begum
Dr.Sonal Panchal
THANK YOU FRIENDS FOR HELPING AND CONTRIBUTING TO THIS PRESENTATION
Thank you ISPAT & INSUOG for all these educational activities during CORONA times

4. Magnitude of Problem
Congenital & Genetic Disorders in India
Disorder Frequency
at birth in stillbirths in NN Deaths
Congenital 2-5% (1:50 , 6 lacs/yr) 13.6 % 7 – 10 %
Malformations
Chromosomal 0.5% ( DS – 1:916 , 22,000 / yr )
Single Gene 0.6% ( B- thal – 1:2700 , 9000/ yr ; SCD - 5,200 /yr ;
DMD + SMA - 4,500 / yr )
( IC Verma , Preventive Genetics ,2006 )

5. Chromosomal defects
Source:
Kagan KO et al. Screening for Chromosomal Abnormalities by First Trimester Combined Screening and Noninvasive Prenatal Testing. Ultraschall in Med 2015; 36:40-46)

9. Routine Antenatal Care 1990s…….
Early scan to
diagnose
pregnancy &
dating
Fetal defects
22-24 wks
Anomaly scan

10. Routine Antenatal Care 2005
11-14 wks
Fetal defects
20-23 wks
P I P I P
The great
Ob syndrome

11. Routine Antenatal Care 2010
11-13+6 wks
Fetal defects
Chemical markers
Major Cardiac defects
Uterine artery Doppler
20-23 wks
Anomaly scan

12. Watch
carefully the
picture

13. Where do all these markers come from?
(adrenals)
(liver)

14. SCREENING PRINCIPLES
Screening is done for:
• Disorders with common prevalence
• Have significant impact on perinatal
mortality & morbidity
Conditions to be fulfilled:
• Accessibility, Availability , Affordability
• Must understand Cost of burden of the disease vs
cost of screening

15. What Do Screening Tests Do?
1. Identify patients “at risk” for a disorder in an
“unsuspected” population
2. “Filters” patients for further diagnostic testing
3. Results are given as
“high risk” or “low risk”
based on a “cut off” value

16. PRENATAL SCREENING
• I TRIMESTER
• History, MAP,
Ut A doppler /
Biochem
• II TRIMESTER(?)
• UT A doppler
• CERVICAL
LENGTH – II
trimester
• I & II
TRIMESTERS
• ULTRASOUND
• BIOCHEMISTRY
• CELL FREE DNA
• I & II TRIMESTERS
ANEUPLOIDY ANOMALY
PRE-
ECLAMPSIA
PRETERM
BIRTH

17. PRENATAL TESTING
SCREENING TEST
•Provides individual risk assessment
•Advantages
• Lowers the number of procedures done for diagnosis
• Lowers the procedure related complications
•Disadvantages
• Non diagnostic
• May miss target
DIAGNOSTIC TEST
•A test that WILL identify a disease or defect
•Advantages
• Definitive
•Disadvantages
• Risk associated with diagnostic procedure/s

18. FOGSI OLD CHECK LIST OF 2009,MODIFIED IN 2017 AT FOGSI T.O.G.
Added in 2020

19. prelude
OFFER SCREENING UNIVERSALLY
PRENATAL SCREENING COUNSELING
• Pretest Non-judgemental counselling
• Limitations / Risk / Benefits / Costs
• Post test counselling
Pt information leaflets / Video / Inforgraphics

20. Maternal Serum Markers
• First Trimester: Papp-A
Free beta hcG
• Second Trimester: AFP
uE3
hCG
Inhibin-A

22. “Life is not free of problems, but happiness is having a path forward”-
 Earlier assessment enables more options and in the case of pre-eclampsia – the
opportunity to prevent.
 Not just aneuploidy, also includes maternal pre-eclampsia and structural defects
 Pregnancy MUST be Ultrasound dated with CRL (ideally 8-10 wks)
 Range of options: from Biochemistry only to a full portfolio, with multiple ultrasound
markers
What is BE HAPPY First Trimester Pregnancy Check?

28. SCREENING FOR ANEUPLOIDY
• Trisomy 21 - Down syndrome - 1 in 800
• Trisomy 18 – Edwards synd. - 1 in 10,000
• Trisomy 13 – Patau’s syndrome > 1 in 10,000
Apriori Risk
MATERNAL AGE,
Previous screening)
Identify
markers
(USG, Biochem)
Modify Risk
Identify
Patients for
invasive testing

29. PART 2- WHAT TO OFFER
BEFORE
14
WEEKS
AFTER
14
WEEKS

30. 30
TEST FIRST TRIMESTER SECOND TRIMESTER
CRL 45-84 BPD 30mm -51mm
10 11 12 13 14 15 16 17 18 19 20
Sequential Screen
NT,
PAPP-A, hCG
+PLGF
AFP, hCG, uE3,
Inhibin A
CONTINGENT
SCREEN
NB/ DV/TR
Serum Integrated
Screen
hCG , PAPP-A AFP, hCG, uE3,
Inhibin A
Quad Screen
E-FTS
PAPP-A, hCG
+AFP +PLGF
AFP, hCG, uE3,
Inhibin A
1
4
w
G
r
a
y
Z
o
n
e
THE TIMELINES FOR SCREENING
NIPS – after 10 weeks – ideal before 17 weeks to help decision making < 20 weeks

31. First trimester screening FOR CHROMOSOMAL ANOMALIES
FETAL
CROWN
FETAL
RUMP
NT/NB
DV
TR

32. First Trimester Screening – The yield
Test Name
Time of
Test(in
Weeks)
Markers
DR(%) FPRs ( % )
Early Biochemistry 9-10^+6 MA+hCGB, PAPP-A
~60-65 5 %
COMBINED FIRST
TRIMESTER
SCREENING
11-13^+6 MA+hCGB, PAPP-A+NT
~90-91 3 %
11-13^+6 MA+hCGB, PAPPA+NT
+NB+tri cuspid flow+facial
angle+Ductus Venoses
~95% 3 %
Penta Screening 10-
13^+6
MA+ hCGB, PAPPA, PIGF,
DIA+NT+NB
98 % 1.2 %
BE HAPPY TEST

33. Screening for Trisomy 21 at 11- 14 weeks
2-stage (contingency) screening- UK system
COMNINED TEST (NT+BIOCHEM) FOR ALL
Fetal NT and
free BhCG and
PAPP-A at 12 wks
High risk
>1:250
low risk
<1:1000
CVS
Reassure
Borderline risk
1 in 251-1:1000
Further
screening
Nasal bone
DV, TR
NIPS

34. 99
NIPS

35. New Screening Protocol for Trisomy 21 + PE + ONTDs
in Indian Scenario
Viability Scan: 6-8 weeks
LOW RISK HIGH RISK
NIPS
Anomaly scan at 19 weeks
with genetic sonogram
Amniocentesis
CVS
LOW RISKHIGH RISKLOW RISK HIGH RISK

36. SO PROPOSAL IS INDIAN CONTINGENT SCREEN
COMBINED OR eF.T.S.
OR
INTEGRATED FIRST AND SECOND TRIMESTER
SCREEN
OR
SCREEN BY N.I.P.S

37. SCREENING FOR PE
• Maternal history
• Maternal blood pressure
• Uterine artery doppler
• Biochemical markers
• Combination ..
PAPP-A / PLGF

38. Pre-Eclampsia
• Adaptive disease when there is
relative placental imbalance
(Utero Placental mismatch)
– Late onset may be beneficial to
the baby – improved quality of
survival in late mild PE
• Early onset may have
implications for mother / baby

39. First trimester screening for Preeclampsia
Maternal serum PIGF,
Mean arterial Blood pressure
- Implantation
- Maternal artery remodelling
Detection rate – 90%
0
10
20
30
Falsepositive(%)
Doppler
12%
30%
PP13
9%
Doppler
& PP13

40. History + MAP + UT A doppler + PLGF & PAPP-A
• 93% detection rate (5% FPR) for early
preeclampsia (<34 weeks)
Poon LC et al Hypertension 2009
A combination of markers at 11-13 weeks is ideal

41. • Low-dose aspirin initiated in early pregnancy is an
efficient method of reducing the incidence of
preeclampsia and IUGR.
(Obstet Gynecol 2010;116:402–14)
Is there intervention??

42. Is there intervention if screened in first trimester?
• Low-dose ASA may prevent as many as 50% of
cases of PE & IUGR when treatment is initiated
before 16 weeks of gestation
Bujold E, et al Obstet Gynecol 2010;116(2 Pt 1):402–14.
Bujold E et al J Obstet Gynaecol Can 2009;31:818–26.

43. First Trimester
Second Trimester
Third Trimester

44. Second Trimester Screening
Screening
Strategy
Time of
Test(in
Weeks)
Markers Used DR(%) FPRs ( %)
Triple Test 15-21^+6 MA+AFP,hCGB, uE3 ~65-70 5-7%
Quadruple
Test
15-21^+6 MA+AFP,hCGB, uE3
,Inhibin-A
~70-75 5-7%
Integrated
Test
15-21^+6 1st Trimester – PAPP-A
11-13+6/7weeks
(11 Wks is Ideal)
2nd Trimester –Quad
AFP, uE3, FBhCG,
Inhibin-A
(15-17 Wks is ideal)
93% 3 %
TRIPLE TEST SHOULD BE GIVEN UP LOW DETECTION RATES
NIPS CAN BE OFFERED AS A SCREENING TEST 10-17 WEEKS

45. Contingent II trimester “Genetic sonogram”
To modify risks after
FTS combined,
Quadruple
Or
Integrated screening
Needs high USG expertise
Software for calculation
ARSA Absent NB

46. When there are major structural defects
NO screening is offered – Direct testing is preferred
IN SUCH CASES NO QUAD TEST: DIRECT AMNIOCENTESIS

47. SCREENING FOR FETAL ANOMALIES- I TRIMESTER
70% detection rate if a systematic scan is done

48. DIAGNOSTIC TESTS
AFTER SCREENING
WHAT TO ORDER ?
AND
WHAT TESTS TELLS WHAT ?

49. CVS
• 11-14 weeks
• Transcervical
• Check for chorionic
villi under
microscope
• Risk of miscarriage
1%
• Need is obviated
now due to NIPT
????

50. Amniocentesis
• 15-18 weeks
• Risk of miscarriage < 1%
• To confirm diagnosis in positive
integrated screen and/or positive
NIPT
• To screen known carriers for
chromosomally abnormal fetus

51. Invasive Diagnostic Techniques
Amniocentesis

52. Testing with foetal cells
Karyotyping
Fluorescence In Situ Hybridization (FISH)
Polymerase Chain Reaction (PCR)
Array‐based comparative genomic hybridization (array CGH) i.e. chromosomal microarray
analysis (CMA) ,next-generation sequencing (NGS) , whole-exome sequencing
Which method to use for Prenatal Diagnosis
Karyotyping?
FISH?
Sequencing?
NGS / CMA?
Shaffer LG, Bui T‐H. 2007. Molecular cytogenetic and rapid aneuploidy detection methods
in prenatal diagnosis. Am J Med Genet Part C Semin Med Genet 145C:87–98.

53. ASSAY GENETIC ABNORMALITY
Karyotyping Detect structural, numerical defects chromosomal Syndromes
FISH syndromes where gene locus is
known
Aneuploidies, Microdeletions,
22q del. , 9;22 in CML
Microarray
Based on RNA
analysis
Deletion and duplication at higher
resolution. Cannot detect
inversions and balanced
translocations
Intellectual disability,
Structural defects, physical/ USG
Biochemical tests
(chromatography
techniques)
Examines proteins (blood, urine ,
CSF) instead of the gene,
metabolite or protein structure.
Disrupts key metabolic pathway
IEM
PCR Detect specific disease-causing
mutations
Triple repeats fragile -X
Molecular testing
•Sanger, NGS,
Exome, Whole
genome
Single gene mutations including
point mutations , deletions ,
duplications within the gene
Holt-oram syndrome (TBX5)
Alagille syndrome(JAG1)
Char syndrome (TFAP2B)
Noonan syndrome (PTPN11)
DIAGNOSTIC
GENETIC TESTING

54. Karyotype can detect
• Some specific abnormalities e.g.
- Trisomies (21,18,13), Klinefelter's syndrome
(XXY and other variations), Triple X syndrome
(XXX)
- Monosomy : Turner syndrome (X0)
• Some Chromosomal deletions
- Cri-du-Chat syndrome (missingCh5)
- Williams syndrome (missing Ch7)
• Translocations : Robertsonian translocations
Limitation : Cannot detect specific gene mutations e.g. cystic fibrosis, Thalassemia.
Resolution limited to around 5 Mb.

55. Molecular cytogenetics :FISH
(Florescence In Situ Hybridization)
• Is a cytogenetic technique used
to detect and localize the
presence or absence of specific
DNA sequences on chromosomes
• Allows visualization of small
region on chromosome too small
to be identified by
karyotyping(submicroscopic
deletion)
• Unlike Karyotype(21days) results
available with in 24 to 72 hrs as
done on interphase nuclei

56. QF-PCR
 has recently entered the field of prenatal diagnosis to
overcome the need to culture fetal cells, allow rapid
diagnosis of some selected chromosomal anomalies.
 has the advantage of being much less expensive and
allowing the simultaneous processing of much larger
number of samples than FISH.
 Can detect mosaicism of about 20–30% and is superior
to both traditional karyotyping and interphase FISH
for the identification of maternal cell contamination
[ Donaghue et al., 2005]

57. QF-PCR: Offerings
QF-PCR BASIC QF-PCR EXTENDED
Coverage • Trisomies :13,18,21
and Gonsomal
Aneuploidies
 Trisomies :13, 18,21, 15,16,22,
Gonosomal Aneuploidies
 20 common microdeletions and
microduplications:
 DiGeorge Syndrome,Langer-
Giedons Syndrome,Smith-
Magnenis Syndrome,Miller-
Dieker Syndrome,Williams-
Beuren Syndrome,Potoscki-
Lupuski Syndrome

58. Microarray Analysis
Can identify chromosomal aneuploidy , large
structure changes as well as submicroscopic
abnormalities that are too small to be
detected by traditional modalities
A DNA microarray is a thin-sized chip that has
been spotted at fixed locations with thousands of
single-stranded DNA fragments corresponding to
various genes of interest
A single microarray may contain 10,000 or more
spots, each containing pieces of DNA from a
different gene

59. Introduction of chromosomal microarray
analysis into prenatal diagnosis
• CMA is now the first-tier genetic diagnostic test for
children and adults with multiple congenital anomalies,
genetic syndromes, and intellectual and developmental
disabilities, diagnostic yield - 15 to 20%
• CMA is recommended as a primary test, by ACOG ,after
USG finding of structural abnormality unless the
abnormality is “strongly suggestive” of a particular
aneuploidy
• CMA suitable for analysis of stillbirth samples & early
miscarriages , it does not require actively dividing cells
Miller DT, Adam MP, Aradhya S, et al. Am J Hum Genet. 2010;86(5):749–64. Hillman SC, McMullan DJ, Hall G, et al. :
Ultrasound Obstet Gynecol. 2013;41(6) ACOG Practice Bulletin,163:2016

60. Next Generation Sequencing(NGS)
• Sequence each of the 3 billion bases in the human
genome multiple times providing an insight into
unexpected DNA variation missed by other methods.
• Can sequence entire genomes or specific areas of
interest, including all 22 000 coding genes (a whole
exome) or small numbers of individual genes
• By providing a base-by-base view of the genome, NGS
can identify single nucleotide variants (SNV), small
structural changes, and balanced translocations
• increasing information while decreasing costs with a
genome-wide view of variation.
Arch Dis Child Educ Pract Ed. 2013 Dec; 98(6): 236–238

61. SEQUENCING
KARYOTYPE NEXT GENERATION
SEQUENCINGMICROARRAY

62. Whole-exome sequencing(WES)
• Congenital abnormalities identified USG , karyotype and
CMA reveal a diagnosis in up to 20–30%, for the
remainder, single-gene tests or gene panels, may be
useful
• CMA and NGS based methods, such as targeted gene-
panel sequencing and, recently, whole-exome
sequencing (WES), has enabled to diagnose more fetal
genetic conditions
• In WES, majority of coding exons, which represent only
2% of the genome but contain 85% of disease-causing
mutations, are sequenced
Lee H, Deignan JL, Dorrani N, et al. : . JAMA. 2014;312(18). Gilissen C, Hehir-Kwa JY, Thung DT, et al. : Nature.
2014;511(7509) Beaulieu CL, Majewski J, Schwartzentruber J, et al. : . Am J Hum Genet. 2014;94(6)

63. Cont--
• The American College of Medical Genetics and
Genomics recommends considering WES when
specific genetic tests available for a phenotype,
including targeted sequencing , have failed to
determine a diagnosis in a fetus with multiple
congenital anomalies suggestive of a genetic
disorder
• Routine use of whole-genome or whole-exome
sequencing is not recommended outside of the
context of clinical trials
ACMG Board of Directors. Genet Med 2012;14:759–61 , ACOG Practice bulletin ,Number 682, December 2016

64. Take home message
SCREEING SHOULD BE OFFERED TO ALL
POPULATION IRRECPECTIVE OF RISK
While
• Testing should be focused on the individual
patient's risks, reproductive goals, and preferences
Patients should understand the benefits and
limitations , including the conditions that will not be
detected by tests
PRE AND POST TEST COUNCELLING IS VERY
IMPORTANT

65. Various Integrated screening in
strategies (1st and 2nd trim)
Main strategies:
• Fully Integrated
• Step-wise sequential
• Contingent screening
1st trimester:
NT, PAPP-A
No risk estimate
2nd trimester:
Fb-hCG, AFP, uE3, (± Inhibin)
Final risk estimate:
All markers
1st trimester:
NT, PAPP-A, Fb-hCG
Risk estimate
Final risk estimate:
All markers
CVS
NIPT
High risk
Low risk
2nd trimester:
Fb-hCG, AFP, uE3, (± Inhibin)
1st trimester:
NT, PAPP-A, Fb-hCG
Risk estimate
2nd trimester:
Fb-hCG, AFP, uE3, (± Inhibin)
Final risk estimate:
All markers
CVS
NIPT
HR
Borderline risk
No further
screening
LR

67. Ultrasound
CVS
Amniocentes
is
NIPT
Combined Test
Other Screening Test

70. Thank you
Screen with the right test at the right time