Index

Sr. No. Topics Page No.

1. Introduction 2

2. Prenatal Screening 4-22

3. Cytogenetics 22-38

4. Newborn Screening 39-41

5. Haemoglobinopathies Screening 42-45

6. NIPT 46-47

7. FAQ’s – NIPT 47-49

8. FAQ’s – PNS 49-63

9. FAQ’S – Cytogenetics 63-70

10. Sample Collection Giudelines 71-73

11. Contacts 74

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 LILAC INSIGHTS PVT LTD
INTRODUCTION

Lilac Insights is India’s only dedicated Fetal genetic screening & diagnostic Test Center which
follows the guidelines of Fetal Medicine Foundation, established under the scientific leadership
of Dr. Prathima Radhakrishnan and Dr. Anita Kaul: India’s leading Fetal Medicine Experts who
are FMF Certified Trainers in India trained by Prof. Kypros Nicolaides who is the founder of FMF,
UK.

The purpose of Lilac Insights is to provide evidence and guideline based Screening and
Diagnostic test solutions from conception till the birth of the baby and even beyond that. Lilac
Insights also focuses on research and facilitation In the field of Genetics and prenatal area.

Lilac Insights is the country's only NABL accredited dedicated Fetal Medicine Lab with facilities
to offer evidence based prenatal screening tests utilizing Fetal Medicine Foundation (FMF) UK
protocols.

Lilac Insights was founded in 2011 with a single minded objective of bringing standardization in
the area of prenatal screening. Within a short span of four years, Lilac Insights has already
become the country's most preferred Fetal Medicine screening & diagnostic service provider
with processing centers at Navi Mumbai, Chandigarh, Ludhiana and branch offices at Pune,
Aurangabad, Delhi, Bangalore, Kolkata, Indore, Chennai & growing. Institutions and Doctors
from more than forty five cities across the country have placed their trust on Lilac Insights for
their Fetal Medicine Investigation needs.

Besides Fetal Medicine Foundation (UK) guideline based prenatal screening, Lilac Insights has
also established, a state of art genetic testing centre scientifically headed by Prof Jayarama
Kadandale, visiting faculty at Sloan Kettering cancer institute, USA.

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Trimesters in Pregnancy:

Trimesters. A normal, full-term pregnancy is 40 weeks, and can range from 37-42 weeks.

It's divided into three trimesters. Each trimester lasts between 12 and 14 weeks, or about three months

First Trimester is between 1st week to 13th Weeks of pregnancy

Second Trimester is between 14th Weeks to 26th Weeks of pregnancy

Third Trimester is between 27th Weeks to 40th Weeks of pregnancy

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 Prenatal Screening
Every couple wants to have a normal baby. Ultrasound investigations can detect only major
physical deformities and very less chromosomal abnormalities. The only tests that can give a
definitive answer whether the baby is affected or unaffected with a major chromosomal
abnormality are invasive (Amniocentesis or CVS) and carry a risk of causing miscarriage.
Therefore, if we do amniocentesis on every pregnancy, we will be causing more abortions of
normal pregnancies than the number of chromosomally abnormal babies we will pick.

Hence, globally the protocol that is followed is as follows:

1) First screen the pregnancy as high risk or low risk through a screening test.
2) Offer invasive tests only to those pregnancies that are high risk. Let the low risk
pregnancies not get subjected to invasive tests.

Goal of Prenatal Screening

To offer a pregnant woman her accurate & personalized risk for most common fetal
aneuploidies so that based on a comparison of this risk with the risk of losing a baby because of
the invasive test, she can chose to accept or reject the invasive test.

Illustration:

Screening test risk > risk of abortion from invasive test= Decision in favor of doing an invasive
test.

Screening test risk < risk of abortion from invasive test = Decision in favor of not doing an
invasive test.

What cut-offs are used for classifying a screening result as high-risk or low-risk?

Cut-off commonly used in India and in many parts of the world to classify a woman as high risk
or low risk is 1:250. This is because studies show that the risk of miscarriage caused by

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amniocentesis is 1:250. This means if amniocentesis is performed in 250 women, 1 will abort.
So if the screening test result gives a risk higher than 1:250 eg. 1:240, then the woman is
considered as having a risk of giving birth to a Down syndrome baby that is higher than the risk
she has of abortion caused by amniocentesis. Naturally she will opt for the lower risk option of
the two which is amniocentesis. Whereas if the screening test results for a woman is lower than
1:250, eg 1: 260, it means the risk she has of giving birth to a Down syndrome baby is lower
than the risk of miscarriage due to amniocentesis. Naturally in such a case, she will opt not to
do an amniocentesis since doing an amniocentesis in this case has a higher risk.

Note: Remember Higher the number is the risk ratio, lower is the risk & Lower the number in the
risk ratio, higher is the risk.

Screening tests are not diagnostic tests

It is wrong to say that a Dual marker, Triple marker or a Quadruple marker is done to detect
Down’s syndrome. Though many clinicians use this statement to describe why they do these
tests, it is important to understand that Dual marker, Triple marker or a Quadruple marker tests
are screening test and not diagnostic tests and hence they cannot detect an abnormality. Tests
that can detect abnormalities are diagnostic tests. Screening tests only give a probability of
having an abnormality. How does knowing the probability of having a problem help? This
probability is important for the woman to take decisions to do or not to do an invasive test.
Doing an unnecessary diagnostic test will expose her fetus to the risk of miscarriage since the
diagnostic test is invasive, on the other hand not doing a diagnostic test where it is warranted
may lead to a chromosomally abnormal baby go undetected and get born causing a lifetime
trauma.

A diagnostic test gives a 100% diagnosis, a yes or no answer, whereas a screening test will
always give a probability (1 in____). A diagnostic test therefore is not associated with false
positive results (meaning the test result wrongly showing a normal case as positive) or false
negative results (meaning the test result wrongly showing an abnormal case as negative). A

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screening test by nature is associated with false positive or false negative. One can only try to
minimize false positives and false negatives by using additional markers and by strict adherence
to standards. For understanding what is a marker, read below.

What is a marker?

Marker is an indicator. For illustration purpose, consider you are walking amongst a crowd of
thousand people and somebody is told to identify you from the crowd from a distance, what
will he do? He will use identifiers or markers that he associates with you like Complexion,
height, style of walking, style of dressing etc. etc. If the person knows you are 6 feet tall
(Marker here being 6 feet height), He will use this marker and try to identify you from the
crowd. What will happen? Out of the crowd of 1000 people, all the people who are 6 feet tall
will get identified as you. Further, if he knows that you have a fair complexion, then he will add
fair complexion as an additional marker to height. Thus he will be able to shortlist all fair
complexion people from the group of 6 feet tall people, he has earlier identified as you. Then
he may add style of dressing as a marker and so on.

What is happening in the above illustration? By adding more markers that are known
characteristic of you, the person trying to identify you from a distance is increasing his chance
of correctly identifying you from the crowd. However, how much ever markers he keeps
adding, he can never say he is 100% sure. At best he can achieve a near 100% surety. He can be
100% sure only when he physically comes in the crowd face to face to you and says “Yes it is
you”. This is the difference between a screening test and a diagnostic test. In screening tests
you can keep adding markers to improve the chance of correct identification of an abnormality
but can never be 100% sure, whereas in diagnostic tests you are 100% sure( the face to face
example).

Amniocentesis/CVS are 100% sure diagnostic tests in which fetal cells are directly viewed
under the microscope for chromosomal abnormalities, whereas Dual/Triple/Quadruple is
screening test.

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Evolution of screening tests

Maternal age: To start with, High Maternal age of more than 35 years was used as a marker to
screen (segregate) pregnancies into high risk and Low risk, which means pregnancies above 35
years were considered as high risk and advised a diagnostic test like CVS or amniocentesis,
while all pregnancies lower than 35 years of age were considered low risk and no further
testing was offered. This was a good approach at that time, since it is known that as age of
pregnancy advances, chances of having a chromosomal problem increases due to the ovary
becoming older. At 35 years the risk of having Down’s syndrome is close to 1:250, therefore
pregnancies above 35 years were considered as having a risk higher than 1:250 and directly
advised a CVS or amniocentesis, whereas pregnancies less than 35 years of age were
considered to have a risk lower than 1:250 and hence no further testing was advised to them.
The disadvantage of this approach was this approach lead to a high rate of miscarriages without
significantly reducing Down syndrome births. Why? Because though it is an established fact that
risk of chromosomal problem like Down’s syndrome increases with age, it is also an established
fact that 80% of pregnancies happen in women less than 35 years of age and

therefore majority of Down’s syndrome births are also happening in

pregnancies less than 35 years. With the maternal age as marker approach, all less than
35 years pregnancies are considered safe and no further testing is offered. This approach is
therefore failing in its purpose of reducing Down’s syndrome child-births. On the other hand by
considering all pregnancies above 35 years as high risk and offering all these pregnancies direct
invasive test, we are no doubt picking up all Down’s syndromes in this age group but at the cost
of causing more miscarriages of normal unaffected pregnancies by exposing all pregnancies
above 35 years to invasive tests. This approach therefore has more risk than benefit and hence
is not a good approach.

The approach of using Maternal age as marker was found to have a detection rate of only 35
% (which means we are able to identify (pick-up) 35% of Down’s syndrome affected
pregnancies) at a False positive rate of 5% (5% False positive rate means of 100 pregnancies
reported as increased risk, 95 were confirmed to be affected with Down’s syndrome on doing

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the diagnostic test, while 5 were found not affected with Down syndrome on diagnostic test
but still had got reported as positive on screening test).

Triple marker: Since maternal age as a screening test was not turning out to be a very
successful screening test, researchers were constantly working on finding out newer and better
markers for Down’s syndrome. On studying hormonal levels of pregnancies born with Down’s
syndrome babies, they found out that those pregnancies that resulted in Down’s syndrome
birth showed different levels of certain hormones in the second trimester (15 weeks to
20weeks 6 days of pregnancies) in comparison to pregnancies that gave birth to a baby not
affected by Down’s syndrome. The observation was those pregnancies that landed up with
Down’s syndrome births showed higher levels of free βhCG (free Beta unit of Human Chorionic
Gonadotropin), lower levels of AFP (Alfa feto-protein) and lower levels of uE3 (unconjugated
estriol) than normal pregnancies. Thus, high free b-hCG, low AFP & low uE3 levels in second
trimester got added as additional markers for Down’s syndrome to Maternal age and a
screening test called Triple marker was conceived, triple because of three hormones being
checked. It is important for you to understand that though typically a Down’s syndrome
affected pregnancies will show a distribution of certain hormones different as above, from
unaffected pregnancies, at times even Down’s syndrome unaffected pregnancies can show
hormonal levels similar to that of a Down syndrome pregnancy due to physiological reasons like
placental insufficiency among others. These pregnancies though unaffected will get reported as
High risk for Down syndrome due to their atypical hormonal distributions. These are the false
positives. Similarly, rarely even Down’s syndrome affected pregnancies may show normal
hormone levels representative of a normal pregnancy and hence get reported as Low risk for
Down syndrome. These are false negatives. This is precisely why these tests are called
screening tests & not diagnostic tests. Triple marker was found to have a detection rate of 60%
at a false positive rate of 5%

Quadruple marker: There was constantly research being conducted to improve the detection
rates of triple marker. It was thereafter discovered that Down’s syndrome affected pregnancies
also show elevated levels of a hormone Dimeric Inhibin-A. Addition of this hormone to triple

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marker improved the detection rates to 70% at a false positive rate of 5%. Though research is
still going on to find more markers and improve detection rates further, Quadruple marker
screening is at the moment the most sensitive and most preferred screening test in the second
trimester, world-wide.

Dual marker: While Quadruple marker offers advantage of improved detection rates, both
triple marker and Quadruple marker have a limitation and that is they can only be performed in
the second trimester i.e. between 15 weeks to 20 weeks 6 days of pregnancy. It is important for
you to know here that the upper limit to abort an abnormal pregnancy is 20 weeks, legally as
well as from mother’s safety point of view. Since a quadruple or triple marker can only be done
after 15 weeks, if the screening test gives a positive (high risk) report, the patient will need
some time to decide on doing the confirmatory amniocentesis test and if the patient decides to
do the amniocentesis test for confirmation, a chromosomal analysis report will take 2-3 weeks.
If the chromosomal report confirms a Down syndrome affected pregnancy, by the time, the
patient gets the report; she may cross 20 weeks, the upper limit to take a decision on aborting
the pregnancy if they decide to. If therefore there is a screening test that offers good detection
rate, low false positive and can be done earlier in pregnancy, it will obviously offer a lot of value
to the patient in terms of time for decision making.

Researchers discovered that free βhCG tend to show elevated levels in Down’s syndrome
pregnancies not only in the second trimester but also in the first trimester i.e. between 11
weeks to 13 weeks 6 days. They also found out that another first trimester hormone called
Pregnancy Associated Plasma Protein (PAPP-A) generally showed lower levels in Down
syndrome affected pregnancies than in Down syndrome not affected pregnancies. Combining
free βhCG& PAPP-A was found to offer a detection rate of 60% at false positive rate of 5% for
Down syndrome. This is the Dual marker test since it checks for two hormones. A strong
advantage of Dual marker is it can be performed in the first trimester of pregnancy and hence
gives a good time to the patient for decision making in the event the results are positive.

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 Free β- PAPP-A AFP uE3 Inhibin-A
hCG
Down’s syndrome (Trisomy 21) Elevated Low Low Low Elevated
Edwards syndrome (Trisomy 18) Low Low Low Low Low
Patau’s syndrome (Trisomy 13) Low Low Low Low Low
Open Neural Tube Defects NA NA Elevated NA NA
Typical Distribution of hormones in Down syndrome, Edwards’s syndrome, Patau's syndrome
& Open Neural Tube defects

Free β- PAPP-A AFP uE3 Inhibin-A

hCG
First Trimester Yes Yes No No No
Second Trimester Yes No Yes Yes Yes
Hormones tested in First Trimester Screening methods and Second Trimester Screening
methods

Combined First Trimester Screening Test: While there was continuous research to discover
newer biochemical markers to improve detection rates, simultaneously there was research
being done to find out ultrasound markers that can be used either alone or in combination with
hormones to improve detection rates further. This led to the discovery of an ultrasound marker
called Nuchal Translucency (NT). Nuchal Translucency is an area behind the neck of the fetus
that is formed due to accumulation of fluid. This is visible on the scan from 11 weeks of
pregnancy, and is most prominent at 12 weeks and then keeps receding as the pregnancy
advances. At 13 weeks 6 days, NT disappears completely and gets converted into Nuchal Fold
Thickness (NFT). Please remember NT is Nuchal Translucency and is different from NFT. Both,
if increased are markers for Down’s syndrome but NT is a marker in the first trimester and
NFT is a marker in the second trimester. Down syndrome affected fetuses generally have
increased Nuchal Translucency measurements in comparison to fetuses not affected with
Down’s syndrome. Hence NT in itself is a marker for Down’s syndrome. In isolation, NT offers a
detection rate of 70% at false positive rate of 5%. However if it is used along with maternal

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age and Dual marker, the overall detection rate improves to 90% at a False positive rate of
5%. This is called the combined test or Combined First Trimester Screening test and is today
the standard screening test in the first trimester across the world.

Figure 1- Nuchal translucency & Nasal Bone

Figure 2- Tricuspid Regurgitation Figure 3- Ductus Venosus

Nasal bone, Tricuspid Regurgitation and Ductus Venosus: As work was continuously and
is continuously being done to identify newer and more accurate markers; either blood markers or
ultrasound markers, after nuchal translucency, nasal bone was discovered as another ultrasound
marker which if added to combined first trimester screening test can improve the detection rate

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further by 1%. Nasal bone is the bone of the nose and Down's syndrome pregnancies generally
don’t have a nasal bone hence, if during the NT scan, if nasal bone is found absent, it is considered
as a pointer towards a Down’s syndrome affected pregnancy. Similarly Tricuspid regurgitation (TG)
and Ductus Venosus (DV) are two more ultrasound markers for Down’s syndrome, each adding to
the detection rate by 1%. If all thee markers are used together (which means Maternal age +NT
+freeBhCG +PAPP-A+ Absent NB +TG+DV), one can achieve a detection rate of 95% at false positive
rate of 5%.

Alternate Screening Models: Combined First Trimester Screening (MA+ NT+ free BhCG+ PAPP-
A) & Quadruple marker screening are the most widely accepted screening models in the first
trimester and second trimester respectively. Various permutations, combinations of the above
screening models have been developed and are practiced in different countries across the
world. Some of these are mentioned below:

Integrated Screening: Integrated Screening is essentially a two stage screening process.

Stage.1 A combined First Trimester Screening is done between 11 and 13 week 6 days. The
results from this are not revealed to the patient and then Stage 2 a quadruple screening test is
performed at 16 weeks. The results from the quadruple screening test are then integrated with
the previous Combined First Trimester Screening results and a consolidated report based on
MA, NT, first trimester hormones and second trimester hormones is issued. Integrated
Screening test offers a detection rate of 95% at 5% false positive rate. However when an
Integrated screening is done, it is important that the patient is given only one final risk after the
second stage has been done and results from the second stage evaluation integrated with the
first stage results. Separate risks from first trimester and second trimester screening should not
be given as this may lead to confusion with the patient getting two risks and not knowing which
one to believe.

Contingent Integrated Screening: An offshoot of the integrated screening test mentioned

above is Contingent Integrated screening. In contingent integrated screening, the first stage
test is done on all patients. Patients who get reported as high risk in this stage are straightaway

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offered confirmatory invasive testing and only those patients who are reported low risk in the
first stage are offered an integrated screening test.

Serum Integrated Screening: In those places where facilities to do NT are not available, a serum
integrated screening test is offered as an option to Integrated screening test. Serum integrated
screening test is same as the integrated screening test except that NT is not included in the
serum integrated screening test. Serum Integrated screening test is PAPP-A+ free B-hCG
assessment in the first trimester integrated with a quadruple assessment in the second
trimester & one consolidated risk assessment report being issued after the quadruple
assessment.

Early biochemistry combined with NT at 12 weeks: The Classical Combined First Trimester
Screening model involves performing both NT and PAPP-A+ free B-hCG on the same day
anytime between 11 weeks to 13 week6 days of pregnancy. This is also called the OSCAR(One
Stop Clinic for Assessment of Risk). It was later found that the hormones give more reliable
results earlier in pregnancy, as early as 10 weeks while NT gives reliable results at 12 weeks.
Hence this model was developed which is an offshoot of combined Screening where hormones
are assessed at 10 weeks (report is not released) & then combined with NT done at 12 weeks.
This gives a detection rate of 91% at a false positive rate of 5%

Contingent first trimester Screening: This is a cost-effective offshoot of the combined first
trimester screening model and a good alternative for those places where NT is not easily
available. In this model, biochemistry (PAPP-A & free B-hCG) is evaluated at 10 weeks and a risk
generated based on this evaluation. For patients whose risk is lower than 1:1500, no further
testing is done, for patient whose risk is higher than 1:65, direct invasive test is done. Only for
the patients whose risk is between 1:65 and 1:1500, an NT is done at 12 weeks, included in the
dual marker evaluation and a combined test report released. This model brings down the need
for NT from 100% of the patients to 20% patients and offers a detection rate of 85% at a 5%
false positive rate.

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Medians & MoMs

In routine pathology, eg checking hemoglobin levels of a patient, the patient’s blood sample is
analyzed for Hb levels and the levels found in the patient’s blood sample are directly reported
along with the reference range that shows the lower normal limit and the upper normal limit. If
the patients Hb levels are within the reference levels, the patients Hb is considered normal, if
the patients measured Hb levels are less than the lower limit of reference levels, the patients
Hb is considered low. Very simple, Isntit !!!

In prenatal screening however, it isn’t that simple. First the patient’s hormone values are
measured. The measured hormone values are then multiplied with the median values for the
hormone to give the Multiple of Median (MoM) Value for the hormone for the patient. These
MoM values are then converted into the risk result. Explanation of how measured values are
different from MoM values is given below:

A median is value is the middle value you get if you arrange a set of numbers in ascending or
descending order. Eg. If the set of numbers are 1, 2, 3, 4, 5, then the median value here is “3”.

Researchers doing research on prenatal screening have developed Median values for different
hormones in first trimester and second trimester and these median values are provided in the
form of a software called Risk assessment software.

Prenatal Screening process follows the following step.

Step.1: Absolute values of the hormones in the blood sample are measured by precision
analyzers.

Step.2: These values are then fed into the software. The software does the following
calculation.

How many times of the median value for the hormone is the measured absolute value of the
patient?

Observed value = Multiple of median (MoM) x Median

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Eg. If the patients absolute value for PAPP-A is 25 and the median value for PAPP-A is 50, then
the Multiple of Median (MoM) for PAPP-A for the patient is 0.5 (25 is what Multiple of the
median value which is 50? Answer is 0.5)

Exercise:

1) If Median value is 50 and patients observed value is 100. What is patients MoM value?
2) If median value is 0.5 and patients observed value is 3. What is patients MoM value?

Very Very Very Very Important

Median values for any hormone eg PAPP-A is -

a) Gestation age specific: Two women of the same age say 25 years, one having a
gestation age of 10 weeks and the other having a gestation age of 11 weeks will have
different Median values and hence different MoMs for the same observed hormone
value.
b) Maternal age specific: Two women of the same gestation say 10 weeks of pregnancy
but one woman being of 25 years of age, the other 35 years of age will have different
median values and hence different MoMs for the same observed hormone value.
c) Ethnicity specific: Two women of the same gestation age, say 12 weeks, but one woman
is Asian & other is African, they will have different median values and hence different
MoMs for the same observed hormone value.

Other factors which influence the Medians and hence the MoMs, are Smoking, Previous history
of Downs, Type of Conception i.e. whether the pregnancy is a natural pregnancy or a case of
assisted reproduction, since ART pregnancies are known to have different hormonal levels in
comparison to naturally conceived pregnancies.

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IVF pregnancies:

IVF pregnancies have different characteristics than naturally conceived pregnancies and hence
different Median values for hormones taking into consideration IVF specific parameters have
been developed. Many infertility cases are due to problem with the functioning of the ovary in
which case egg (ovum) is taken from a donor woman. Since most infertility cases are woman of
advanced age who have tried all possibilities to conceive, have been unsuccessful and then
resorted to donor eggs and the donors that are chosen are younger woman with healthy
ovaries, maternal age to be taken into consideration for risk assessment is of the source of egg.
Taking the fertilized woman’s age can give a falsely raised risk since advanced age is a marker
for Down’s syndrome. Many women are also administered hCG injections to sustain
pregnancies. Since free β-hCG is one of the hormones that is measured during first trimester as
well as second trimester screening, if the woman has been administered an hCG injection just
before collecting the sample for prenatal screening, screening results will tend to show high
b-hCG levels due to the external hCG administered resulting in false positives.

It is a common practice in order to improve the success rate of IVF techniques to implant
multiple embryos, so that at least one embryo will successfully grow. The disadvantage of this
approach, however, is that many times, it results in multiple pregnancies like triplets,
quadruplets etc. In such situations many couples decide to selectively abort extra fetuses
retaining the healthiest fetus. In such situations, if blood sample is collected immediately after
the fetal reduction, hormones secreted from the aborted fetus will falsely raise the levels in the
maternal circulation causing erroneous results. It is recommended in such cases to give at least
4 weeks gap after the fetal reduction and then collect the sample so that the hormones
secreted by the dead fetus has washed out of the maternal circulation. Sometimes a
pregnancy starts as a twin however one fetus being abnormal or unhealthy gets naturally
aborted leaving only one fetus. This is called a case of Vanishing twin and is nature’s own way
of selecting healthy pregnancies to progress. Same precautions are to be taken in cases of
vanishing twins as in Fetal reductions.

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Screening in Twin Pregnancies:

There are two types of twins 1) Dichorionic & 2) Monochorionic.

Monochorionic twins are identical twins and are known to arise from the same zygote
(monozygotic). Being from the same zygote, they have the same genetic make-up which means

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if one fetus is affected with Down’s syndrome, the other fetus will also be affected. Dichorionic
twins are however those twins which arise out of two different zygotes and hence it isn’t
necessary that if one fetus is affected with a chromosomal abnormality, the other fetus will also
be affected. In twin pregnancies, the hormones found in the mother’s blood will be a pool of
hormones contributed by both the fetuses. Since in prenatal screening, we are analyzing
hormones from the mothers blood, it is not possible in the event one fetus is affected with
Down’s syndrome while the other is not, it is not possible to segregate what proportion of
hormones have been secreted by the affected fetus and how much by the unaffected fetus. It is
therefore not possible to give discriminatory risk assessments through blood hormone based
screening tests. Hence a Dual/Triple/Quadruple marker alone is not recommended in case of a
dichorionic pregnancy. It can however be done if Nuchal Translucency measurements are
available because affected fetus will tend to have an increased NT and unaffected fetus will
tend to have a lesser NT. By combining these different NT measurements of the two fetuses
with a common biochemistry, one can give two different risk assessments for the two fetuses.
Triple/Quadruple marker however should not be done in dichorionic twin pregnancies since
there is no discriminatory marker like NT available for doing triple/quadruple marker.

In monochorionic twin pregnancy a Dual/triple/quadruple can be performed with or without NT

measurement since whatever risk assessment is done is a common risk for both the fetuses,
their genetic make-up being similar.

Effect of temperature:

hCG (human chorionic gonadotrophin) is a temperature sensitive hormone. It is known that

hCG which is what the body secretes keeps getting dissociated into its alpha and beta subunits
and it is the beta subunit that is measured in dual/triple/quadruple marker assessment.
Exposure of blood sample to high temperatures (more than 20 degree C) for prolonged period
of time is known to increase the rate of dissociation of the alpha and beta subunits of hCG
causing increased levels of free B-hCG to be measured than the actual levels at the time of
blood draw, resulting in increased false positives.

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By ensuring proper cold-chain i.e. storing samples in refrigerator door compartment after
collection, transporting with frozen ice gel packs and also minimizing the transportation time,
one can ensure false positives are controlled. Alternatively one can use Dry blood spots for
Dual markers. Dry blood spot method stabilizes free B-hCG and prevents false positive rates
from increasing. Dry blood spots however cannot be used for Triple/Quadruple marker or
integrated screening.

Ultrasound & Blood Tests Are Part of Prenatal Screening

Nuchal Translucency

 Ultrasound Examination includes measurement of Nuchal Translucency (NT) – Thickness of

fluid behind the neck of baby.
 Nuchal Translucency observed in all fetuses between 11 weeks to 13 Weeks 6 Days.
 In a fetus with Trisomy 21 or other abnormalities, the size of Nuchal Translucency tends to
be higher than in normal fetuses.
 Carrying out a Nuchal translucency measurement by appropriately trained Sonologists
(Certified by Fetal medicine Foundation or Matching the criteria’s of NHS on NT) is very
much important to achieve accuracy in measurement & that criteria’s are;
o Image Magnification should be such that the fetal head and upper thorax occupy
the whole screen.
o Fetal position – Midline saggital section of the fetus should be obtained & fetus
should be horizontal on the screen, either supine or prone.
o Care must be taken to distinguish between fetal skin and amnion.
o The fetus should be in Neutral Position, with the head in line of spine.
o The widest part of the translucency must always be measured.
o The umbilical cord may be around the fetal neck in about 5% of cases, in such cases
it is more appropriate to use the average of the two measurements.
o Caliper Placement - Measurement should be taken with the inner border of the
horizontal line of the calipers placed on the line that defines the NT thickness.

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 Blood Tests

A small amount of blood is taken from the pregnant woman’s arm. It is sent to the Program.
At different times during pregnancy, her blood is tested for substances such as:

 PAPP-A (Pregnancy Associated Plasma Protein A)

 hCG (Human Chorionic Gonadotropin)
 AFP (Alpha-Fetoprotein)
 uE3 (Unconjugated Estriol)
 Inhibin-A (DIA)

Above Biochemical’s are tested in an appropriate time to take out more information about
fetus’ status towards aneuploidy which is as follows;

 Dual Marker/Double Marker/ First trimester Screening (10 weeks - 13 weeks 6 Days)
o Nuchal Translucency (NT) – 11 Weeks to 13 Weeks 6 Days
o PAPP-A (Pregnancy Associated Plasma Protein A)
o Free Beta - hCG (Human Chorionic Gonadotropin)

 Triple Marker (15 Weeks-20 Weeks 6 Days)

o AFP (Alpha-Fetoprotein)
o Free Beta - hCG (Human Chorionic Gonadotropin)
o uE3 (Unconjugated Estriol)

 Quadruple Marker (15 Weeks-20 Weeks 6 Days)

o AFP (Alpha-Fetoprotein)
o Free Beta - hCG (Human Chorionic Gonadotropin)
o uE3 (Unconjugated Estriol)
o Inhibin-A (DIA)

20
 ONTD & Abdominal Wall Defect Screening(16 Weeks – 18 Weeks)
o AFP (Alpha-Fetoprotein)

 Integrated Screening/Serum Integrated Screening

o Dual Marker + Triple/Quadruple Marker

Important Abbreviations and meanings

NT: Nuchal Translucency (Accumulation of fluid behind the neck of the fetus seen between 11
weeks to 13 weeks 6 days of pregnancy)

CRL: Crown Rump length (Length of the fetus from crown to rump. Considered the most
accurate Ultrasound dating parameter)

GA: Gestation Age (Age of pregnancy generally expressed in week)

LMP: Last Menstrual Period (First day of the last menstrual period, the woman had)

BPD: Biparietal Diameter (Ultrasound measurement used to determine the gestation age in the
second trimester)

FL: Femur Length (Ultrasound measurement used to determine the gestation age in the second
trimester)

HC: Head circumference (Ultrasound measurement used to determine the gestation age in the
second trimester)

EDD: Expected Date of delivery

FTS: First Trimester screening, commonly referred to as Combined Screening.

STS: Second Trimester Screening

Quad: Quadruple screening

DR: Detection rate

FPR: False Positive Rate

21
PPV: Positive Predictive Value also called OAPR (Odds of being affected given a positive risk). It
is the ratio of true positives to total positives and is a determinant of how effective a screening
test is. Higher the PPV or OAPR, more sensitive is the test.

Cytogenetics
The study of chromosomes, their structure and their inheritance is referred to as Cytogenetics.

What are chromosomes?

Genes are encoded in the DNA that makes up a number of rod shaped organelles called
chromosomes in the nucleus of each cell. Chromosomes contain the DNA (the genetic
information) that facilitates all the aspects of what makes a human being a functional organism.

In humans, the normal cell contains 46 chromosomes, made up of 22 pairs of autosomes and a
single pair of sex chromosomes, XX in normal females and XY for normal males.

One member of each of these pairs is derived from each parent.

Chromosomes consist of supercoils of DNA, which is tightly coiled to give a rod shaped
structure. Members of a pair of chromosomes are described as homologous chromosomes
which carry matching genetic information i.e. they have same genes in the same sequence.

22
How chromosomes look?

Chromosomes are visualized during the cell division which appears as thread-like structures
under the light microscope. All chromosomes have a short arm and long arm separated by
the centromere. The short arm is designated as p and the long arm as q.

Normal Chromosomes

23 Pairs of Chromosomes = Total 46 chromosomes in a cell

22 Pairs are called as Autosomes and 1 pair is called as Sex Chromosomes.

This is called the diploid/euploid number. Females carry two X chromosomes (46,XX) while
males have an X and a Y (46,XY). Germ cells/gametes (egg and sperm) have 23 chromosomes:
one copy of each autosome plus a single sex chromosome. This is referred to as
the haploid number. One chromosome from each autosomal pair plus one sex chromosome is
inherited from each parent. Mothers can contribute only an X chromosome to their children
while fathers can contribute either X or Y.

Abnormalities of chromosome number or structure, which are usually clinically significant, can
arise in either in the somatic cells or germline cells by errors in cell division.

23
Karyotype

Karyotype refers to the standard chromosome set of an individual. Cytogenetic analyses are
done on chromosome preparations that have been treated and stained to produce a banding
pattern specific to each chromosome. This allows for the detection of subtle changes in
chromosome structure.

Following microscopic analysis, either photographic or computerized digital images of the best
quality metaphase cells are made. Each chromosome can then be arranged in pairs according to
size and banding pattern into a karyotype. The karyotype allows the cytogeneticist to even
more closely examine each chromosome for structural changes. A written description of the
karyotype which defines the chromosome analysis is then made.

Normal Female: 46, (XX)

24
Normal Male 46 XY

Chromosome Abnormalities

Although chromosome abnormalities can be very complex there are two basic
types: numerical and structural. Both types can occur simultaneously or individually.

1) Numerical abnormalities

i) Aneuploidy:

An abnormal number of chromosome such as having a single extra chromosome

(47) or a missing chromosome (45)

Trisomy: Having one extra Chromosome

Example:

-Trisomy 21 (Downs Syndrome): 47,XX (+21)/47,XY (+21)

-Trisomy 18 (Edwards Syndrome): 47,XX (+18)/47,XY (+18)
-Trisomy 13 (Patau Syndrome): 47,XX (+13)/47,XY (+13)

25
 Monosomy: Having one missing Chromosome

Example:

- Monosomy X (Turners Syndrome): 45, X

ii) Ploidy Changes:

Having addition of complete set of 23 chromosomes

Example: Triploidy (69 Chromosomes)

Tetraploidy (92 chromosomes)

Basically, numerical changes involve the loss and/or gain of a whole chromosome or
chromosomes and can include both autosomes and sex chromosomes. Generally chromosome
loss has a greater effect on an individual than does chromosome gain although these can also
have severe consequences. Cells which have lost a chromosome are monosomy for that
chromosome while those with an extra chromosome show trisomy for the chromosome
involved. Nearly all autosomal monosomies die shortly after conception and only a few
trisomy conditions survive to full term. The most common autosomal numerical abnormality is
Down’s syndrome or trisomy-21. Trisomies for chromosomes 13 and 18 may also survive to
birth but are more severely affected than individuals with Down Syndrome. Another general
rule is that loss or gain of an autosome has more severe consequences than loss or gain of a
sex chromosome. The most common sex chromosome abnormality is monosomy of the X
chromosome (45,X) or Turner Syndrome. Another fairly common example is Klinefelter
Syndrome (47,XXY). Although there is substantial variation within each syndrome, affected
individuals often lead fairly normal lives.

Occasionally an individual carries an extra chromosome which can't be identified by its banding
pattern, these are called marker chromosomes.

26
2. Structural abnormalities involve changes in the structure of one or more chromosomes.
They can be incredibly complex but for the purposes of this discussion we will focus on the
three of the more common types:

1. Deletions involve loss of material from a single chromosome. The effects are typically
severe since there is a loss of genetic material.

2. Duplications: A portion of the chromosome is duplicated, resulting in extra genetic

material.

3. Inversions occur when there are two breaks within a single chromosome and the broken
segment flips 180° (inverts) and reattaches to form a chromosome that is structurally out-
of-sequence. There is usually no risk for problems to an individual if the inversion is
of familial origin (has been inherited from a parent.) There is a slightly increased risk if it is
a de novo (new) mutation due possibly to an interruption of a key gene sequence. Although
an inversion carrier may be completely normal, they are at a slightly increased risk for
producing a chromosomally unbalanced embryo. This is because an inverted chromosome
has difficulty pairing with its normal homolog during meiosis, which can result in gametes
containing unbalanced derivative chromosomes if an unequal cross-over event occurs.

27
4. Translocations - : A portion of one chromosome is transferred to another chromosome.
There are two main types of translocation.

i) Reciprocal Translocation

Breaking and Exchange between 2 chromosomes

ii) Robertsonian Translocation

Breaking and Exchange between 2 Acrocentric Chromosomes ( Acrocentric
Chromosomes: 13,14,15, 21 & 22)

In Acrocentric Chromosomes, P arm is non-functional and it is very small.

28
5. Ring Chromosome:
A portion of a chromosome has broken off and formed a circle or ring. This can happen with
or without loss of genetic material.

6. Isochromosome:
Two p-arms attach to same centriole & two q-arms attach to same centriole,
thus de-activating the chromosome.

7. Mosaicism:- Mosaicism is when a person has 2 or more genetically different sets of cells
in his or her body. These people are called mosaics and in the vast majority of these cases
the abnormal cell line has a numerical chromosome abnormality. Structural mosaics
are extremely rare. The degree to which an individual is clinically affected usually depends
on the percentage of abnormal cells. A routine Cytogenetic analysis typically includes the
examination of at least 15-20 cells in order to rule out any clinically significant mosaicism.

29
These are just some of the more common abnormalities encountered by a Cytogenetic
Laboratory because the number of abnormal possibilities is almost infinite.

Mosaicism can be Structural as well as Numerical.

30
Examples of Chromosome Abnormalities

Some examples of numerical and structural chromosome abnormalities:

Example 1: A Classical Down Syndrome case. Abnormal male karyotype with trisomy for
chromosome 21(three copies of chromosome 21) – 47,XY,+21.

31
Example 2: An inversion in chromosome 9. Normal karyotype with inversion in
chromosome 9-46, inv (9) (p11q13). It is a normal variation and does not have any clinical
consequences.

32
Example 3: An unbalanced robertsonian translocation between chromosomes 14 and 21.
Abnormal karyotype with unbalanced robertsonian translocation between chromosome 14
and 21-46,t(14;21)(q10;q10)+21.

33
Example 4: A balanced reciprocal translocation between chromosomes 5 and 16.
Karyotype with balanced reciprocal translocation between long arm of chromosome 5 and
short arm of chromosome 16-46,t(5;16)(q15;p13.1).

34
Example 5: A balanced Robertsonian translocation between chromosomes 13 and 14.

Karyotype with balanced robertsonian translocation between chromosome 13 and

chromosome 14-46,t(13;14)(q10:q10).

35
Fluorescent IN SITU Hybridization (FISH)

Fluorescent IN SITU Hybridization (FISH) is a relatively new technology utilizing fluorescently

labeled DNA probes to detect or confirm gene or chromosome abnormalities that are generally
beyond the resolution of routine Cytogenetics.

How it works?

The sample DNA is first denatured, a process that separates the complimentary strands within
the DNA double helix structure. The fluorescently labeled probe of interest is then added to the
denatured sample mixture and hybridizes with the sample DNA at the target site as
it reanneals (or reforms itself) back into a double helix. The probe signal can then be seen
through a fluorescent microscope and the sample DNA scored for the presence or absence of
the signal.

Interphase FISH: FISH can be used in interphase cells to determine the chromosome number of
one or more chromosomes as well as to detect some specific chromosome rearrangements
that are characteristic for certain cancers. The primary advantage of interphase FISH is that it
can be performed very rapidly if necessary, usually within 24 hours, because cell growth is not
required.

A good example is the Aneuploidy test which is performed on amniotic fluid cells or CVS cells
when there is a strong clinical indication for one of the common trisomies. The sample nuclei
are denatured and hybridized with DNA probes for chromosomes 13, 18, 21, X, and Y and
results usually obtained within 24 hours. Routine Cytogenetics is included with an Aneuploidy
test to confirm the results or detect any abnormalities not detected by interphase FISH.

36
Figure:- Normal Chromosome 18 (Aqua Blue Colour)

37
Sample Collection SOP:

 POC samples have to be sent in transport media provided by Lilac Insights.

 POC’s often contain large numbers of bacteria, so for a short period it is fine to keep the
specimen at room temperature. For overnight or longer, it is better to place the
specimen in the refrigerator to keep the bacteria from overgrowing the specimen.

 Specimen type

o Placenta – Tissue that contains chorionic villi

o Umbilical Cord

o Skin – Take biopsy from under the arm or inner thigh, Avoid muscle, tendons or

tough skin (such as heel)

 Specimen Requirements

o Specimen requirements will be 3-4mm POC Specimen or 50-100 mg each tissue

o If possible always include chorionic villi.

o Do not Place in formalin

o Do not freeze

 Whole Fetus samples, internal organ or part of organ samples are not accepted.

38
 Newborn Screening:
 Newborn screening is a health program designed to screen infants shortly after birth for
a list of conditions that are treatable but can cause serious damage to babies’ health.
 Newborn Screening is performed to screen Congenital & Heritable Disorders (Metabolic
disorders and inborn errors of Metabolism).
 These disorders may cause severe mental retardation, illness, or death if not treated
early in life
 If treated, infants may live relatively normal lives
 If Untreated, Can Results in;
o Growth problems
o Developmental delays
o Behavioral/emotional problems
o Deafness or blindness
o Retardation
o Seizures
o Coma, sometimes leading to death

 Common Disorders Screen in Newborn Screening;

o Congenital Hypothyroidism
o Congenital Adrenal Hyperplasia
o G6PD
o Galactosemia
o Cystic Fibrosis
o Biotinidase deficiency
o Haemoglobinopathies
 Sickle Cell Anemia
 Sickle C – Disease
 S-Beta Thalassemia
 Variant Hb

o IEM’s (Inborn Error of Metabolism) – 45 Disorders

 Fatty Acid Oxidation Disorder
 Organic Acid Disorders
 Amino Acid Disorders

39
 Panels for NBS

Sr No. Category Parameters

Congenital Hypothyroidism (TSH)
Essential NBS
1.
Congenital Adrenal Hyperplasia – 17 OHP

Congenital Hypothyroidism (TSH)

Congenital Adrenal Hyperplasia – 17 OHP

Galactosemia
Basic NBS (Bio 4)
2.
G6PD

Cystic Fibrosis

Biotinidase Deficiency

IEMs (45 Disorders)

IEM’s (TMS)  Fatty Acid Oxidation disorders
3.
 Organic Acid Disorders
 Amino Acid Disorders

4. Expanded NBS (FS ) Basic NBS + IEM’s

Hemoglobinopathies
 Sickle Cell Anemia
5. Haemoglobinopathies  Sickle-C Disease
 S-Beta Thalassemia
 Variant Hb

40
 When to get Newborn Screening done?

 The first screen at 24-48 hours or before leaving hospital, whichever is

first
 It can be performed between 2-20 days of birth
 Premature and low weight babies are required to have screens in same
time frame

Why Lilac Insights for Newborn Screening???

 Center accredited by CDC (Atlanta) for Newborn Screening

 Customized Newborn screening panels developed for the Indian
population based on ICMR Newborn screening project findings.
 Advance US FDA Approved technology – Time resolved fluoro-
immunoassay offers higher sensitivity than conventional ELISA.
 Scientific Support from Metabolic Experts.

41
Haemoglobinopathies (Thalassemia)Screening:

 Thalassemia is a genetic disorder caused due to mutation which leads to abnormal

structure of one of the globin chains of the hemoglobin molecule.

 Thalassaemia is an autosomal recessive disorder which passed down through families

and is carried on a recessive gene.

 When genes are mutated it means that they are permanently altered, so thalassemia is
a lifelong condition.

 The most common types of haemoglobinopathies are;

o Alpha thalassemia causes due to the mutation in the alpha haemoglobin chains
o Beta thalassemia causes due to the mutation in the beta haemoglobin chains.
Beta thalassemia major is life threatening disorder.
o Hb-S Trait: Blood disorder in which there is a single amino acid substitution in the
hemoglobin protein of the red blood cells, which causes these cells to assume a
sickle shape, especially when under low oxygen tension.
o Hb-D Trait: One of the variant of Haemoglobin cause mild hemolytic anemia and
mild to moderate splenomegaly mostly found in northern part of India.

Prevalence of Thalassemia:

 It is estimated that 1.5% of the world population are carriers of beta thalassemia and
about 50% of the incidence is from the south East Asian population which mainly
includes our country, India.

 The carrier rate for beta thalassemia is an average of 3.2% of the population i.e. an
estimated 4 crores of the population are carriers of beta thalassemia. However the

42
 distribution of the beta thalassemia gene is not very uniform in India and the prevalence
is very high among certain communities.

Why get tested?

 If any couples are planning to have children and both partner carry the trait for
thalassemia, then future children could be born with thalassemia disease, which is a
serious medical condition so Prenatal and other testing options are available to couples
found to be at risk for having a baby with the disease.
 If one partner carry the trait for thalassemia, but other partner does not, then child
could inherit the trait from them. It is important that this goes into their medical records
for children and grandchildren’s future family planning.
 The child affected with beta thalassemia major occurs at 6 months of age. The affected
infant fails to thrive and become progressively pale. Feeding problems, diarrhoea,
irritability, recurrent bouts of fever and progressive enlargement of abdomen may
occur. The child will require periodic blood transfusion and drug treatment for the rest
of his life. Barring bone marrow transplantation, there is no cure available for beta
thalassemia and thus the need arises for diagnosis during pregnancy.

43
44
How get tested?

Thalassemia Testing
in Pregnancy

Thalassemia Screening
(HB Electrophoresis)
Female Partner

Positive Negative

Thalassemia Screening
(Hb Electrophoresis) Routine follow up
Male Partner

Positive Negative

Documentation of one
Mutation analysis of Both
partner carrier & continue
partners
with Routine follow up

Prenatal Diagnosis of
Identified Mutations

45
InsighT/ InsighT Plus (NIPT-Non Invasive Prenatal Testing):

 During pregnancy, fragments of fetal DNA are released in maternal blood circulation.

NIPT analyzes this cell-free fetal DNA to check certain chromosomal conditions – mainly

Trisomy 21 (Down Syndrome), Trisomy 18 (Edward Syndrome) or Trisomy 13 (Patau

Syndrome) which is performed on maternal peripheral blood.

 DNA is extracted and purified from the mother’s blood sample and analysed via whole

genome shotgun sequencing method using Next generation sequencing (NGS)

technology that allows the most comprehensive screening. Sequencing data are

thoroughly analysed by enhanced bioinformatics, to deliver clear & reliable test results.

 It can be offered after 10 weeks in the pregnancy for Singleton Pregnancy and 12

weeks for Twin Pregnancy.

 This test has a sensitivity and specificity of over 99% for the common chromosomal

disorders listed above, which makes it a highly accurate and reliable screening test

 Prenatal cell-free DNA screening is much more sensitive and specific than traditional

first and second trimester screening, such as the first trimester screening and the quad

screen. As a result, prenatal cell-free DNA screening can often help women who have

certain risk factors avoid invasive testing that carries a slight risk of miscarriage,

including amniocentesis and chorionic villus sampling (CVS).

 Prenatal cell-free DNA screening doesn't screen for all chromosomal or genetic

conditions. A negative test result does not ensure an unaffected pregnancy.

46
 This does not provide information on physical defects, such as cardiac or brain

abnormalities and spina bifida, or fetal growth so it is therefore advisable that patient

still have ultrasound scans at 11-13 weeks and at 18-20 weeks to examine the fetal

anatomy, and at 30-32 weeks to examine the fetal growth.

 In few cases, the test may not give result. This is due to technical reasons (Low fetal

fraction or hemolysis) which do not suggest that there is a problem with the baby so

before undergoing prenatal cell-free DNA screening (NIPT), health care provider or a

genetic counselor will explain the possible outcomes and what they might mean for you

and your baby. Be sure to discuss any questions or concerns you have about the testing

process.

 The reporting of results varies depending on the lab. Results might be reported as

screen positive or screen negative, high risk or low risk for abnormality, or as a

probability.

InsighT FAQ’s

1. How & when to do the NIPT Test?

 Doctor needs to advice this test. The test would entail a simple procedure of
drawing blood from your body. A collection kit with a special tube will be available at
Lilac Insights.

 Test can be advised preferably after 10 weeks of gestation for singleton pregnancy &
after 12 weeks of gestation for Twin pregnancy.

2. Under what circumstance would a re-sampling be required for NIPT?

47
  In few cases, the test may not give a result. This is due to technical reasons with the
analysis of that particular sample and does not suggest that there is a problem with
the baby.

 If they are not able to retrieve a fetal fraction of >4% from the maternal peripheral
blood.

 If sample is hemolysed by the time it reaches the lab.

 In this situation, the test will be repeated with redrawing of your blood sample at no
extra cost.

 After 2 attempts if test will not be able to give results due to any technical reasons,
in such rare situations, a refund will be made to patient/Customer.

3. What would the result show?

 If the NIPT test shows a high risk for Trisomy 21 or 18 or13, it does not mean that
the fetus definitely has one of these defects. If you want to be certain that the fetus
has one of these defects you should get CVS or Amnio done.

 If the NIPT test shows that there is a low risk (less than 1 in 10000) that the fetus has
Trisomy 21 or 18 or 13, it is unlikely that the fetus has one of these defects.

4. What is the reliability of NIPT Test?

 NIPT is highly accurate screening test with an accuracy of 99% & screen only
aneuploidies of 21, 18, 13 & sex chromosomes.

 It does not provide information on other rare chromosomal abnormalities.

 It does not provide information on physical defects, such as heart or brain

abnormalities and spina bifida or fetal growth.

5. What if the test comes screen positive/Screen Negative?

 NIPT is highly accurate screening test with an accuracy of 99% so if the result is
positive, then a diagnostic procedure should be considered for confirmation.

 If the result comes negative that does not mean that the baby is unaffected as its
highly accurate screening.

 Confirmation can only be possible through CVS & Amniocentesis.

48
 6. What are the things require requesting NIPT?

 Collection Kit containing Streck Tube, Requisition form & Patient Consent form.

 Dr. Recommendation Letter

 Xerox of recent Ultrasound report

 Patient Identity proof (Xerox of PAN Card, Aadhar Card etc.)

PNS FAQ’s
1) What is the purpose of PNS? Why to be done for every pregnancy?

 The purpose of PNS is to provide expectant parents with information to make informed choices
and decisions

 PNS can only screen common genetic disorders (aneuploidies) such as Downs syndrome,
Edwards syndrome, Patau syndrome and Neural tube defects. It is not a test to detect these
disorders, it is a screening test done for pregnant women which divides them into a high risk or
a low risk category.

 Every pregnant woman should be screened because these genetic abnormalities are sometimes
known to be associated with family history but mostly occur due to random gamete formation
during fertilization. Hence, every pregnancy is at a risk for common trisomies (most common
being trisomy 21)

2) What are the tests offered in PNS?

 Double/Dual Marker (First trimester screening)

 Triple Marker (Second trimester screening)

 Quadruple Marker (Second trimester screening)

 Integrated Screening (Combined FTS and STS)

49
3) What is the current practice (in general) of PNS?

Currently the awareness of prenatal screening is not well established. There are many
organizations or set of doctors who refer Prenatal Screening on indications like high maternal
ages, previous histories like missed abortions or babies born with specific genetic disorders or in
case of abnormal sonography reports.

The awareness of which screening program should be offered is also not clear.

FMF certified sonologists and some well informed OBG’s: Dual marker with NT scan (some
prefer Integrated Screening)

Other OBG’s and radiologists: Quadruple marker or Triple marker

4) How are the results of the tests given?

The results are generally clearly indicated as Low risk or High risk. In case the evaluated risk is
between the cut-off (1:250) to 1:1000 then a second trimester scan along with a genetic
sonogram is recommended.

5) What is low risk and high risk?

Low risk indicates the patient is above the cut-off risk and may not need further testing, which
essentially states that accordingly to the data analyzed from the software for the particular
patient her values are above the cut-off risk and the likelihood of having a baby with Downs (or
other aneuploidies tested in screening) is very rare (the possibility cannot be denied because
this is not a diagnostic test)

High risk indicates that the patient is below the cut-off risk and will definitely need further
prenatal diagnostic testing in order to rule out the possibility of chromosomal disorders.

50
6) What does false positive mean

Prenatal screening
result

TRUE HIGH RISK FALSE

POSITIVE POSITIVE

Chromosomal Chromosomal
disorder T21, disorder not
T13, T18 identified
identified by
cytogenetics

7) What does false negative mean?

Prenatal Screening

FALSE TRUE
LOW RISK
NEGATIVE NEGATIVE

Chromosomal Chromosomal
disorder T21, T13, disorder not
T18 identified identified
bcytogenetics

51
8) Which is the specific biochemical markers checked for in different prenatal screening tests?

 Dual Marker : PAPP-A + beta-HCG

 Triple Marker : beta-HCG +AFP +uE3

 Quadruple Marker : beta-HCG +AFP +uE3 + Inhibin-A

9) Which are the details collected with a PNS sample? Why are the details
Important?
The mandatory details to be collected with a sample are weight of the patient, DOB, Smoking
Status, Ethnicity, Previous History of Trisomies, Scan details, Conception, If patient is on HCG
injections and Pregnancy type (Single/twins) and date of blood collection.

DOB to calculate age risk.

Weight of the patients- it’s a correction factor for calculating MOM’s

Smoking Status - it’s a correction factor for calculating MOM’s

Ethnicity - it’s a correction factor for calculating MOM’s

Previous history of Aneuploidies- it’s a correction factor for calculating risk

Scan details- important to calculate the exact gestation age as well as risk assessment.

Conception type - it’s a correction factor for calculating risk

If patient is on Beta HCG injections- if patient is on HCG injection then a sample should be
collected after 48 hrs of injection. Reason being person taking HCG injections could have altered
values in risk assessment if sample collected before 48hrs.

Date of collection - it’s used for calculating gestation age.

10) Which sonography markers are used to calculate gestation?

CRL, BPD, HC and corrected EDD are the sonography markers used to calculate Gestational age.
CRL is measured in the first trimester. BPD and HC are measured in the second trimester.

11) What is an intermediate risk? What do we offer in such cases?

When the adjusted risk of a prenatal screening report falls between 1:250 – 1:1000, the patient
is mentioned to be in intermediate risk.

52
 If it’s a double marker report which mentions intermediate risk, the patient is advised to do an
integrated screening where a second trimester screening (either triple or quadruple marker)
would be asked for, the adjusted risk of the second trimester screening would be combined with
the adjusted risk of the first trimester screening and a final risk would be given. The detection
rate of which is 90-92%.

If the second trimester screening report mentions intermediate risk, the patient could be either
directly asked for a direct prenatal diagnosis, a detailed anomaly or genetic sonogram.

12) Why is maintaining temp imp during sample transport?

Temperature variations would result in high beta hcg and inhibin values which could result into
false positive results, hence it is important to maintain ambient temperature during transport.
Whole blood samples should reach the lab before 48 hrs and serum sample should reach the lab
before 72 hours. Measurements of beta hcg and inhibin collected beyond these time limits are
unreliable.

13) Why are serum samples better to whole blood?

Serum sample have a better shelf life than whole blood. Whole blood samples can hemolyse.

14) What does the hospital staff need to be trained about sample transport and TRF entry?

Samples not transported according to the sop’s could result into spillages or wrong results.
Incomplete TRFs could result in delay of TAT.

15) What is our TAT for FTS and QST (Both for Mumbai and territories)?

TAT for FTS is 48 hrs and for QST is 72 hrs (please note TAT is from when the samples is received
in the lab)

16) Explain Auto Delfia and Roche principle?

Auto delfia uses time resolved fluro immune assay. Roche uses chemi Luminescence.

53
17) Which softwares we use?

We use ASTRIA for FMF certified doctors and lifecycle for non FMF certified doctors.

18) Why don’t we use prisca?

Lilac insights follow the FMF protocol for prenatal screening. As per FMF guidelines we can use
Astria software for risk assessment for patient who has done the NT from FMF certified
sonologists.

We are also using Lifecycle software for the biochemistry based risk assessment which has
designed by Professor Howard Cuckle (One of the member from fetal medicine foundation)

Prisca is not accredited by fetal medicine foundation so we can’t use it for prenatal risk
assessment.

19) Why Gel Vacutainer preferred than Plain Bulb?

Gel tube vacutainer offers better sample integrity compared to a plain tube in reference to
serum separation and hemolysis prevention.

20) What are the sample rejection criteria for PNS?

 Sample collected in an expired tube or wrong tube

 If sample is hemolysed of lipemic

 Insufficient sample

 Sample received after 3 days of collection.

 Sample received before 10 weeks, between 14 to 15 weeks and after 20.6 weeks.

 Sample received for STS in twin pregnancy

21) What are the conditions for asking repeat sample for PNS?

Re-sample is asked for when

 Sample collected in an expired tube or wrong tube

54
  If sample is hemolysed of lipemic

 Insufficient sample

 Sample received after 3 days of collection.

 In case the biochemical markers show higher values, for reconfirmation repeat sample is
asked for.

 In case of Vanishing or fetal reduction, if sample came less than 4 weeks time of procedure

 If patient is on HCG and sample came less than 48 hours of HCG intake.

22) Why is your TRF so exhaustive?

TRF is designed as per the FMF guidelines. Every detail enquired plays a crucial role in
interpretation of risk evaluation.

23) Is old sonography report acceptable for Triple/quadruple marker? If yes how old?

Yes old sonography report mentioning the crl, corrected edd which are useful in calculating the
gestational age can be used.

24) Where to store the blood sample after collection?

Is to be stored in the refrigerator in door compartment (2-8 degree celcius)

25) What to do with sample if it's collected late evening & pick up is not possible?

Is to be stored in the refrigerator in door compartment (2-8 degree celcius)

26) Is it mandatory to collect the blood on the same day that sonography is done?

No it is not mandatory to collect blood on the same day as the sonography but there could be
case scenarios where we have to ensure the window periods like for Dual Marker (10-13.6
weeks) & Triple/Quadruple Marker (15-20.6 weeks)

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27) Why is PNS gestation age specific?

Prenatal Screening involves determining the hormone levels in a pregnant patient & comparing
them with hormone levels typically expected in the pregnancy of similar period (gestation age).
Since hormone levels at different gestation age (days/weeks) in pregnancy are different, proper
comparison can be done only if we know the exact day of the pregnancy that is being
investigated so that we can compare the hormone levels observed for pregnancy of a particular
gestation age with established levels for that gestation age.

28) Why is PNS required to be done only in a particular window period of pregnancy?

Hormones assessed in the first trimester like PAPP-A & Free B-hCG are most sensitive in a
particular period of pregnancy which is between 10 weeks to 13 weeks 6 days. Similarly
hormones that are checked in the second trimester are most sensitive between 15 weeks to 20
weeks 6 days of pregnancy. Secondly the expected hormone levels explained in the first answer
(called medians) have been determined by studies for a particular window period in which the
hormones are found to be more sensitive. Hence if you check for hormones outside the window
period, you will not have median levels of hormones to compare with.

29) Which are the biochemical markers checked for in PNS? What is their significance?

The following biochemical markers are checked for in PNS:

First Trimester (10 weeks to 13 weeks 6 days)

PAPP-A (Pregnancy associated plasma protein A): PAPP-A levels lower than half of expected
PAPP-A levels is a marker for T.21, T.18 and T.13.

Free B-hCG: Free B-hCG(Human Chorionic gonadotropin) levels higher by 2 times the expected
free B-hCG levels is a marker for T.21 while low free B-hCG levels are markers for T.18 and T.13.

Second Trimester (15 weeks to 20 weeks 6 days)

Free B-hCG: Same as above

AFP(Alpha Feto Protein): Increased AFP levels is a marker for Open Neural Tube Defects while
lower AFP levels is a marker for T.21, T.13 & T.18

uE3(Unconjugated Estriol): Low uE3 levels is a marker for T.21, T.13 & T.18

Inhibin-A: Increased levels of Inhibin-A is a marker for T.21.

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30) Why Lilac Insights for Prenatal Screening?

PNS involves determining accurately, levels of certain hormones in a pregnant woman and
comparing it with median levels for the hormones for the same gestation age to generate risk
assessments for the pregnant woman for certain common chromosomal abnormalities like
Down syndrome. In order to be able to get accurate risk estimates, it is important that the
median levels with which the patient levels are compared are accurate and based on extensive
study of pregnant women. The study should be extensive, should factor for difference in
hormone levels due to demographic differences like pregnant woman’s age, Gestation age,
weight, smoking, ethnicity, conception type (natural or Assisted) since each of these factors
cause a difference in hormone levels than the typically expected levels. These are called
difference in medians. Lilac Insights uses medians developed by Fetal Medicine Foundation after
years of extensive research and validated through different multicentric studies and trials. Lilac
Insights also uses reagents and instruments that have been tested and certified by the Fetal
Medicine Foundation & also get its results authenticated through external audit by United
Kingdom External Quality Assurance Scheme (UKNEQAS). This protocol diligence ensures that
results that are generated in the form of risk estimates from Lilac Insights are in accordance with
global standards & assist the doctors with a high degree of reliability in basing their decisions on
offering or not offering invasive definitive tests to patients, both being critical decisions.

31) What is the protocol followed by LILAC INSIGHTS for PNS?

Lilac Insights follows the Fetal Medicine Foundation (UK) protocol for PNS

32) What is FETAL MEDICINE FOUNDATION UK?

Fetal Medicine Foundation is a UK based registered charity working towards developing

standards and providing training in the area of Fetal Medicine. It was founded by Prof
KyprosNicolaides who is also the director for ultrasound at Fetal Medicine Foundation (FMF).
Prof Kevin Spencer is the director of Biochemistry at FMF.

33) Why do we follow FMF protocol?

The protocol developed by the Fetal Medicine Foundation (UK) is based on years of extensive
research conducted by the Fetal Medicine Foundation & validated through studies. This gives an
assurance that the risk assessments that are generated by utilizing this protocol are based on
evidence and hence accurate.

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34) Why is FTS better than STS?

FTS (First Trimester Screening) combines an ultrasound marker called Nuchal Translucency (NT)
& two hormones (PAPP-A & Free B-hCG). NT is in isolation itself is a strong marker for Downs
syndrome and offers a sensitivity of 70%, when combines with PAPP-A & free B-hCG, it offers a
sensitivity (detection rate) of 90%. STS (Second Trimester Screening) is based on only
assessment of hormones and does not have any ultrasound marker. Hence the sensitivity of STS
is 70% for Quadruple and 60% for Triple marker.

Secondly since FTS is done early in pregnancy (between 11 weeks to 13 week 6 days), it offers
early information in comparison to STS. This allows the patient more time to make decisions
regarding invasive testing and making an informed choice in case invasive test results are
positive. Since the medico-legal limit for termination of pregnancy is 20 weeks, STS by virtue of
its late window period does not give sufficient time to the patient to make an informed choice.

35) Why is Quadruple screening test better than Triple screening test?

Every additional marker that is checked improves the sensitivity or accuracy of the Screening
test. QST (Quadruple Screening test) evaluates the risk of the pregnant woman based on four
hormones (free B-hCG, UE3, AFP, Inhibin-A) whereas TST (Triple screening test) evaluates the
risk of the pregnant woman based on three hormones ( free B-hCG, UE3, AFP). QST is therefore
more sensitive (70% detection rate) than TST (60% detection rate).

36) Why do we prefer NT from NT certified sonologists?

Nuchal Translucency is measurement of fluid accumulation at the back of the fetus neck. This is
a very strong marker and most prominent between 11 weeks & 13 week 6 days of pregnancy.
Through extensive studies, likelihood ratios have been developed for Nuchal Translucency
measurements. Likelihood ratio is nothing but the possibility of having a Down syndrome
pregnancy. Which means when we say the likelihood ratio of Downs syndrome in a particular
pregnancy is 10, it means the pregnancy is at 10 time’s higher risk of having a Downs syndrome
pregnancy in comparison to any pregnancy of that age. It is important to understand that these
likelihood ratios for Nuchal Translucency measurements are Crown Rump Length specific which
means LR for an NT of 1.2 at a CRL of 45 will be different than the LR for the same NT at a CRL of
55 and therefore same NT value will give different risks for the same patient at different CRLs.

Technically, any measurement of the fluid at the back of the fetus neck done between 11 weeks
to 13 weeks 6 days, irrespective of the profile of the fetus can be called an NT measurement,
however since in prenatal screening, risks are generated by the risk assessment software that
have pre-set likelihood ratios that have been developed by measuring the fetus in a particular
profile ( image magnification, mid-saggital view, neutral position, on to on caliper placement

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 etc), it is important that for a patient who is being assessed, the NT/CRL measurement should be
done with the fetus in the same profile. Else we will be using over-estimated or under-estimated
NT values for a particular CRL and hence inappropriate likelihood ratios applied by the software
will generate erroneous risk assessments.

We therefore either accept NT measurements from those sonologists who are NT certified by
Fetal Medicine Foundation or we insist on the NT image being sent to us so that we audit the NT
image for being in accordance to the FMF/ISUOG/AIUM guidelines.

37) Why we ask for images from non NT certified sonologist ?

Same as answer for question 10.

38) How are we different to the routine lab?

A routine pathology investigation eg Hemoglobin measurement from two different pathology

labs is expected to give same results, since routine pathology investigation involves just
measuring the patients biochemical levels and comparing it with reference levels (upper & lower
normal limits). In prenatal screening besides measuring the patients biochemical levels, it
involves comparing the levels with appropriate medians which will apply appropriate LRs of the
hormones to generate biochemical values in Multiple of Median (MoM) terms and then
translating the MoMs into Risk estimates for the patients. Accuracy of risk results therefore
depends upon the protocol being employed by the lab. Labs employing different protocols will
give different risks due to different median values.

Lilac Insights strictly follows FMF (UK) protocols which have been developed over years of
extensive research which includes using FMF accredited reagents, FMF accredited instruments,
Medians developed by FMF through the Astraia risk assessment software. Lilac also gets its
results audited externally through the United Kingdom Quality Assurance Scheme (UKNEQAS).

The other difference between Lilac Insights and other pathology labs is Lilac Insights being a
dedicated prenatal screening centre has a multidisciplinary team of countrys leading experts in
the area of Genetics & Fetal Medicine besides an FMF theory certified biochemistry team to
assist the doctors interpret the risk assessment reports appropriately and holistically and to take
what-next decisions.

39) What is integrated screening? When can it be offered?

Integrated Screening is a two- stage screening test developed by Prof Nicholas Wald. In the first
stage, Combined First Trimester Screening (PAPP-A & Nuchal Translucency) is done between 11
weeks -13 week 6 days & results are withheld till 16 weeks. In stage two (16 weeks to 19 weeks),

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 sample is collected and analysed for the quadruple marker. Final risk assessment results are
generated only after the second stage test i.e after integrating the quadruple measurements
with the Combined First Trimester Screening measurements. It is important to note however
that Integrated screening is not the same as doing a FTS separately and STS separately and one
should therefore avoid giving two different risk assessments results, one after the FTS and other
after the STS since this will cause confusion & difficulty in counselling based on two different risk
estimates.

40) What is our post-test support system?

Lilac Insights being a dedicated FMF(UK) accredited prenatal screening centre, has a
multidisciplinary team of experts in the area of Fetal Medicine, & Prenatal genetic who are the
best in the country and trained at the Fetal Medicine Foundation under Prof Nicolaides. These
experts are always available to provide assistance either in interpreting the risk assessment
reports or to provide support in taking what-next decisions after a prenatal screening report has
been delivered.

41) In twins which tests we offer? What details we need? Some labs offer STS, why
Don’t we do it?

We offer FTS/STS/QST in monochorionic twins and only FTS provided the NT has been measured
by a certified sonologist, in Dichorionic twins.

In Twins, we need chorionicity details in addition to the usual patient information.

There are two kinds of twins, Monochorionic and Dichorionic. Monochorionic twins are
considered to have developed from the same zygote and hence are considered to have the
same genetic make-up. Dichorionic twins on the other hand are considered to have developed
from different zygote and hence having different genetic make-up. In twin pregnancies, the
hormones in the maternal blood circulation will be contributed by both fetuses. In a dichorionic
twin pregnancy , where there is a possibility of one fetus being affected with Downs syndrome
and the other being unaffected, it is not possible to determine, what proportion of hormones
have been contributed by the affected fetus and the unaffected fetus and hence it is not
possible to give a separate fetus specific risk assessment unless we have a fetus specific NT
measurement provided. In monochorionic pregnancy however where it is assumed that if one
fetus is affected, the other fetus will also be affected and hence contribution of hormones by
both fetuses in the mothers blood circulation is the same, risk assessment can be done by
averaging the overall hormone measurements from the mothers blood. Sensitivity of a prenatal
screening test in twin pregnancy however is lower than the sensitivity in a singleton pregnancy.

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42) For maternal age more than 35 why do we ask for a direct prenatal diagnosis?

Why is screening not preferred for such patients?

Incorrect question, Screening should be done for all pregnancies irrespective of age.

43) If second trimester screening shows risk for ONTD what is advised?

High levels of AFP > 2.5 MoMs is a marker for Open Neural Tube Defects. If STS shows ONTD, a
detailed anomaly/level-2 scan is advised since all ONTDs get picked up in an anomaly scan
performed as per standards.

44) Please explain fetal reductions and criteria for sample collection in case of fetal
reductions?

Many times especially in assisted reproduction techniques (ART), in order to improve the
success rate of pregnancy outcomes, multiple embryos are implanted so that even if some
embryos fail to grow, atleast one embryo grows successfully. The disadvantage is at times
multiple embryos start growing resulting in pregnancies of multiple orders like triplets,
quadruplets. In such times, the couple is given the option of reducing fetuses to twin or single.
This is done by selecting the most healthiest fetuses as per Ultrasound findings and reducing the
others by injecting Potassium Chloride in the heart of the others. Problem in performing
prenatal screening in a case of fetal reduction is that the hormones contributed by the fetus that
has been reduced will show raised levels of hormones leading to False positive ( as a result of
high free B-hCG) or False negative (as a result of high PAPP-A) results. In Fetal reduction cases
therefore, it is important that the blood sample is collected after a minimum gap of 4 weeks
from the day of fetal reduction, so that effect of hormones secreted by the reduced fetus is
avoided.

Sales Calls FAQs

45) What should be explained to a dr in the first visit?

In the first visit, you should introduce Lilac Insights to the doctor as the country’s only dedicated
Fetal medicine Centre offering Prenatal Screening as per Fetal medicine foundation (UK)
guidelines. If the doctor looks involved & inquisitive, go on to explain the doctor how prenatal
screening is different from routine pathology test as explained in the answer to question 12
above & how Lilac Insights by following FMF guidelines delivers reliable results.

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46) Some doctors only offer PNS in case of high NT or high maternal age?

PAPP-A, free B-hCG& NT are markers independent of each other, hence it does happen in some
cases that despite low NT, the fetus is detected with Downs syndrome. Adding markers will
improve the detection and also reduce incidence of missing Down’s syndrome. PNS therefore
like NT should be offered to all pregnancies and not only to those with high NT. From the
perspective of high maternal age, 80% of pregnancies happening in the world are from below 35
year maternal age women and therefore majority of Downs syndrome births are happening
from women less than 25 years of age. Screening only those women who are above 35 years
means we will be screening a limited population where lesser Downs syndrome births are
happening and not screening the wider population where majority of Downs syndrome births
are happening.

47) I am doing sonography for last 15 years and nobody has questioned that my NT is bad, so why
I should do FMF course?

Sir, we definitely cannot question your expertise in doing NT since you are an expert, our only
request is since we follow FMF protocols for doing First Trimester Screening, we are constrained
by the protocols that we use to accept NTs that have been measured as per FMF protocols since
the Likelihood Ratio’s of NTs that we have in our software are for NTs measured as per FMF
guidelines which are the same as the ISUOG(International Society of Ultrasound in Obstetrics &
Gynecology) or AIUM(American Institute of Ultrasound in Medicine) NT measurement
guidelines.

Sir, the FMF course is free of cost and you can do the course sitting at home. We don’t insist you
do the FMF course but if you do it, there are certain benefits.

 You will get the First Trimester Risk Assessment software license free of cost.

 As a second step to NT certification, you can do the Uterine Artery Doppler certification which
will enable us to perform a comprehensive risk evaluation for your patients which include
screening for pre-eclampsia & Fetal Growth Restrictions besides aneuploidies.

48) I advise my patient marker test and they get tested by their local labs?

Sir, you will appreciate that you will be taking some very critical decisions like advising or not
advising an invasive test to the patient based on the risks provided to you by the prenatal
screening report. On one hand you will not want to advise an unnecessary invasive test to a
patient who is at a low risk, while on the other hand, you will not want a patient to deliver
Downs syndrome baby just because he was not offered an invasive test as the screening report

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 showed a low risk. It is therefore important to check with the lab offering prenatal screening
test, what protocols they use, and if the protocols have been validated through studies. If the
local lab you get your PNS tested from uses evidence based protocols like the ones developed by
Fetal medicine foundation, please continue. If however it doesn’t, we at Lilac Insights offer you
the option of getting your patients prenatal screening done in accordance with the FMF (UK)
protocols. We believe, by doing so, you will experience improved performance from your
prenatal screening program.

49) Difference between Lilac Insights and Perkin: I am sending to perkin, why should I send it to you?

Sir, the aspect of a screening program that has the most pronounced influence on screening
results are the medians and the medians are provided by the risk assessment software. Lilac
Insights is the only prenatal screening centre in the country that utilizes medians developed by
the Fetal Medicine foundation besides using FMF accredited reagents and instruments and
getting the results audited by UKNEQAS.

50) Why should I follow FMF?

Sir, FMF has the largest body of studies and research on prenatal screening based on which it
has developed protocols for prenatal screening. These protocols have now been accepted and
made part of guidelines by virtually every scientific group or association in the area of prenatal
diagnosis. Following the FMF standards will give you an assurance that the risk estimates you
are getting for your patients are in line with global standards.

51) I am comfortable with my existing lab; I have never faced any issue till now, so
why should I go with you?

Same as answer for question 38.

CYTOGENETICS FAQ’s
1) What is Cytogenetics (in general description, chromosome Structures, types of
Chromosomes, normal karytotype)

Cytogenetics is the study of chromosomes (structural and numerical) and their role in heredity.

In humans, the normal cell contains 46 chromosomes, made up of 22 pairs of autosomes and a
single pair of sex chromosomes, XX in normal females and XY for normal males.

On an individual level, the short arm of the chromosome is labeled the p arm, while the long
arm is labeled the q arm. The connection point of the two arms is called the centromere.

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2) When is a cytogenetic study recommended?

There are some common indications for different specialties.

OBSTETRICIANS OR GYNECOLOGISTS

 Amenorrhea
 Prolonged Infertility
 Male infertility
 Bad Obstetric History or Recurrent Abortions
 High risk prenatal screening result
 Family history of genetic disorders
 Hereditary Breast and Ovarian Cancer

PAEDIATRICIANS

 Failure to thrive
 Dysmorphology
 Ambiguous genitalia
 Down Syndrome
 Mental retardation
 Delayed milestones and growth retardation
 Multiple congenital defects
 Edward syndrome
 Patau syndrome
 Klinefelter Syndrome

RADIOLOGISTS

 Abnormal ultrasound findings in pregnancy

 IUFD
 IUGR
 Oligohydramnios/ Polyhydramnios

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3) What are the sample types?

Peripheral blood, Chorionic Villus/Villi, Amniotic fluid, Fetal Cord Blood, Products of Conception

4) What does a Karyotyping report say?

A karyotyping report analyses the structure and number of chromosomes present in the human
cell. It helps to determine if there are any chromosomal abnormalities or rearrangements which
might have any effect on the phenotype.

5) What all can a Cytogenetic report identify? What can a cytogenetic study not identify?
Can a Karyotype report miss any chromosomal abnormality?

A karyotype can identify structural and numerical abnormalities in chromosomes.

It cannot identify single gene disorders (Molecular and Biochemical genetic disorders)

Chromosomal analysis can miss cryptic changes, low level mosaicism (<10%), maternal cell
contamination (CVS, POC) and sub microscopic deletions.

6) What are the different types of chromosomal abnormalities?

Numerical abnormalities: Monosomy, Trisomy, Tetrasomy, Triploidy, Tetraploidy (these are the
common one’s)

Structural abnormalities: Balanced translocations (reciprocal and robertsonian) Unbalanced

translocations, Deletion, Addition, Duplication, Inversions, Ring chromosome.

7) What are the common indications for cytogenetic studies (sample specific)?

Peripheral blood: Amenorrhea, Downs, Lack of sexual characters, Infertility, BOH

AF/CVS/Fetal Cord Blood: Abnormal screening reports, abnormal ultrasound scan, advanced
maternal age, previous family history of chromosomal disorders (could be previous baby or any
other next of kin), parents with chromosomal rearrangements

POC: IUFD, congenital malformations, USG anomalies.

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 8) What is the use of doing KT on POC? How it will help my patients for future pregnancy?

First trimester abortions (unexplained and recurrent) are mostly related to genetic disorders
(chromosomal abnormalities). If we get a chromosomal analysis by KT, it will help in identifying
the recurrence risk and will also help in management of future pregnancies.

9) What are the different prenatal sampling procedures? How are they done and what are are
the risks involved?

Chorionic villus sampling (CVS), Amniocentesis and Fetal cord blood sampling.

CVS is done between 11-13 weeks of gestation.

Amniocentesis is done between 16-20 weeks.

Cord blood sampling is done between 18-20 weeks.

10) What is PCPNDT Act? Why is it important to collect prenatal samples from a doctor

Who has a PCPNDT certificate?

PC-PNCDT stands for Pre-Conception and Pre-Natal Diagnostic Techniques Act

This act has been put in place to stop female feticide. Hence, all prenatal sampling procedures
and tests conducted on those samples are covered in this act. Cytogenetic analysis analyses sex
chromosomes as well and according to this act revealing those sex chromosomes in the report is
illegal.

The doctor conducting the procedure must hold a valid PCPNDT certificate if he is sending
samples for prenatal diagnosis in order to abide by the rules of the act and not promote sex
determination in any way. We have a PCPNDT license for the laboratory and need to get the
necessary documentation from the doctor to perform the test on any prenatal diagnostic
sample. Without these forms the prenatal sample will not be accepted by the lab for any
prenatal diagnostic test.

11) Can a sonologist perform prenatal diagnosis?

Yes if he has a PC PNDT license (certificate).

12) Why is amnio preferred to cvs?

One limitation of karyotyping is that it cannot rule out maternal cell contamination completely.
If there is any abnormality other than the common genetic disorders screened, in a CVS
karyotyping the possibility of placental contamination cannot be ruled out. Hence, any atypical
abnormality found in the CVS karyotyping must be confirmed with an amniotic fluid karyotyping
according to the ISCN guidelines which have be followed.

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13) When can an amniocentesis be done beyond 20 weeks?

If there are any ultrasound abnormalities detected in the fetus beyond 20 weeks an
amniocentesis can be performed. However, while collecting the sample we must explain to the
patient that termination on the basis of the report will not be possible. According to the PC-
PNDT act termination of the fetus beyond 20 weeks is not permitted (is illegal).

14) Explain FISH principle? Which samples can be used in a FISH Study?

Fluorescence In-Situ Hybridization also known as FISH is a laboratory technique used for
detecting and locating a specific DNA sequence on a chromosome. The technique relies on
exposing chromosomes to a small DNA sequence called a probe that has a fluorescent molecule
attached to it. It is mainly used to diagnose specific aneuploidies and sub-microscopic deletions.

15) Can only fish be offered on prenatal samples? What limitation does only fish test have?

No, only a FISH cannot be offered for PND samples because the test only determines specific
numerical changes in the chromosomes. It does not detect structural chromosomal
abnormalities and any numerical abnormality other than 13, 18, 21, X and Y. It can be used to
rule out polyploidies if done for all 5 probes.

16) Why does a normal/abnormal fish report, have to be followed by a karyotype?

FISH is done only for specific chromosomes and can rule out only numerical abnormalities. If
there is a down syndrome detected by a FISH analysis then the finding out the type of down’s
syndrome (numerical or structural) is very important in order to determine the recurrence risk
in the next pregnancy.

17) Can FISH be offered on PB and POC?

Yes, because submicroscopic deletions cannot be seen in a chromosomal analysis. This is why a
FISH is required in order to detect other disorders which might be indication specific (especially
in pediatric patients)

18) What are micro deletions?

Chromosomal deletions smaller than 5 million base pairs (5 Mb)spanning several genes that are
too small to be detected by conventional cytogenetic methods or high resolution karyotyping (2-
5 Mb). Detection of these microdeletions is done by FISH.

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 E.g.: Di-George syndrome, Praderwilli syndrome, Angelman syndrome, William syndrome,
Miller-Dieker syndrome etc.

19) Why is blood for karyotype collected in sodium heparin?

Sodium heparin prevents clotting of the sample which is a prerequisite to obtain lymphocytes
necessary for chromosomal analysis.

20) Why CVS and POC sample collected in media?

Media is a nutrient supplement with balanced salt solutions that prevent dehydration of the tissue
which is necessary for preserving live/viable cells and additional antibiotics which prevent any sort
of contamination.

21) Why is Amniotic fluid sample not collected in media?

Amniotic fluid is a fetal origin fluid which is rich in proteins, carbohydrates and other amino acids,
hence additional supplement is not required.

22) Which forms and requisition details are required for Karyotype?

Form G- Duly filled signed and seal of the doctor which is patient consent necessary for prenatal
samples and Form F according to the laid PCPNDT Act Rules and Regulations.

Other important details:

Name, Age, Sex, LMP or gestational age in case of prenatal sample.

Referral reason and referring doctor, any other patient clinical history.

23) Why is our prenatal TAT 14-21, some labs offer reports in 10 days?

According to the standard guidelines, prenatal TAT is around 14-21 days since the culturing of the
cells take approx. 9-10 days and further around 4-5 days for final analysis and report.

24) Is it important to maintain temperature during prenatal sample transportation?

Yes. Ambient temperature should be maintained to obtain viable cells as high temperatures will
lead to dead cells which in turn will be difficult in growing such samples and in turn delay in final
report or sometimes fail to grow leading to culture failure.

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25) Some Dr’s send Amniotic fluid/CVS samples in syringes, is it ok?

No, since such invasive procedures will be easily contaminated and also lead to spillage of the
sample. As these are non-repetitive samples such practices should be minimised or avoided.

26) When can a sample be rejected (Peripheral Blood, Amniotic fluid, CVS)

 Clotted/ expired/ hemolyzed PB samples

 AF/ CVS generally cannot reject as repeat sampling may incur risk for the patient.

27) How do we rule out maternal cell contamination (MCC) in POC and CVS?

Conventional KT or FISH cannot rule out maternal cell contamination.

In order to rule out MCC, we will require mother’s blood and can run PCR.

28) TRF for cytogenetic tests is unavailable, patient history on letter head of Dr is sufficient?

Generally any sample without TRF will not be entertained. In case of non-availability of TRF, letter
head along with all the necessary details like pt. Name, age, referral reason, gender (in case of
newborn babies sample) and consent form in case of prenatal sample is mandatory.

29) What are specific indications for Product of Conception (POC)?

The Most common indications for POC are repeated missed abortions, BOH, Recurrent
pregnancy loss, Non viable pregnancies, Abnormal anomaly scans, Primi Gravida with missed
abortion, Cystic hygroma, Oligohydramnios.

30) Which samples should be collected? {Please give gestation specific examples}

The collection of sample in sterile conditions, placental tissue with clean villi, fetal skin from
armpit, thighs and finger bits, cord sample of the fetal origin.

In recurrent miscarriage cases send only the following specimen

 Chorionic Villi 25-30mg (Early Gestation)
 Bits of Placental Tissue (25-30mg)
 Soft skin tissue 25-30mg (Late Gestation)
 Cord tissue is also preferred
 Cord blood from fetal end (preferable 2-3ml in EDTA tubes Non-Expired.)

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31) When can a POC sample be rejected?

If the sample is received in formalin then it can be rejected directly. Also if the sample is highly
contaminated with extremely pungent smell or if there is no presence of suitable tissue.

32) Explain POC sample collection and transportation?

POC sample should be collected in the transport media provided by the lab and it should be
transported in the MKT kit. If transportation may take longer times then the sample should be
refrigerated.

33) Why do we not prefer POC sample in normal saline?

Sending the POC samples in normal saline increases the risk of bacterial and fungal
contamination. Also the sample becomes smelly if stored for longer time. Saline affects the
quality of sample, thereby degrading the DNA and the viability of tissue.

34) How do we rule out maternal cell contamination in POC and CVS?

Maternal cell contamination can be avoided by proper cleaning of the sample and selecting the
appropriate tissue, thereby avoiding the red blood cells of maternal origin.

35) What if a POC sample is collected on Sunday, how should it be stored and transported till

It reaches the lab?

If a POC sample is collected on Sunday then the sample should be stored in refrigerator until the
shipment. Do not freeze.

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 LILAC COMMITMENT

SAMPLE COLLECTION GUIDELINES

1. PRENATAL SCREENING

(First Trimester and Second Trimester Screening)

 Sample type: Maternal peripheral blood – 3 ml

 Sample collection tube: Non-ExpiredSerum separation tube (yellow top) / Plain tube
(red top)

 Sample once collected should be sent to the lab or kept in the door compartment of the
refrigerator until picked up by the logistics team/courier. Please DO NOT FREEZE the
sample

 If centrifugation facility is available, kindly centrifuge the sample first (to separate serum)
and then store in the refrigerator, if not please store as it is in the serum clot activator
tube

 The test requisition form (TRF) needs to be filled out with complete patient details;

 Date of Birth
 Weight
 Conception type
 Latest ultrasound report (NT scan in case of first trimester screening)
 Previous pregnancy or child affect with chromosomal disorders
 LMP

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SAMPLE COLLECTION GUIDELINES
2. CYTOGENETICS

(Prenatal and Postnatal)

TEST SAMPLE COLLECTION COLLECTION TUBE( VOLUME

TYPE MEDIA Non Expired)
FISH Amniotic - Sterile falcon tubes 20 ml
and Fluid (Conical bottom)
Karyotyping CVS Sterile transport Sterile falcon tubes 15 – 25 mgs
media (RPMI)
Peripheral - Sodium Heparin 3 – 4 ml
Blood (green top)
Fetal Cord - Sodium Heparin 3 – 4 ml
Blood (green top)
Products of Sterile transport Sterile container -
conception media
PoC specimen should be sent in
the CVS media shown in the
picture

PoC material exposed to

fixatives like Formalin are not
acceptable.

Frozen samples of PoC are not

Amniotic fluid CVS Blood acceptable

 All kits for prenatal diagnostic samples will be provided by Lilac Insights Pvt. Ltd.

 The kit contains TRF, Consent form (Form G), sterile tubes for sample collection and parafilm
(seal) to seal the tubes after sample has been collected

 Sample once collected should be sent to the lab in the boxes/kits provided or kept in the door
compartment of the refrigerator until picked up by the logistics team/courier. Please DO NOT
FREEZE the sample

 The patient consent form (Form G) and TRF for prenatal cases must be duly filled with the
PCPNDT registration number and stamp of the clinician and seal of the hospital/clinic where
procedure has been performed. These are requirements from the PCPNDT which all genetic
laboratories have to adhere by

 Acceptable Specimen for PoC:Placental tissue, Fetal cord blood, Soft tissue ( Ear pinna, Butt
skin, Tip of Finger.

 Unacceptable Specimen for PoC: Whole Fetus, Tissues received in unsterile conditions

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SAMPLE COLLECTION GUIDELINES
3. MOLECULAR GENETICS
(Prenatal and Postnatal)

TEST SAMPLE COLLECTION COLLECTION VOLUME

TYPE MEDIA TUBE
( Non-Expired)
Single gene Amniotic - Sterile falcon tubes 20 mls
disorders, Fluid (Conical bottom)
Microarray, CVS Sterile transport Sterile falcon tubes 15 – 25
Exome media (RPMI) mgs
sequencing, Peripheral - EDTA 3 – 4 ml
Molecular Blood (purple top)
Karyotyping Fetal Cord - EDTA 3 – 4 ml
(MKT) Blood (purple top)
Products of Sterile transport Sterile container -
conception media

Amniotic fluid CVS Blood

 All kits for prenatal diagnostic samples will be provided by Lilac Insights Pvt. Ltd.

 The kit contains TRF, Consent form (Form G), sterile tubes for sample collection and parafilm
(seal) to seal the tubes after sample has been collected

 Sample once collected should be sent to the lab in the boxes/kits provided or kept in the door
compartment of the refrigerator until picked up by the logistics team/courier. Please DO NOT
FREEZE the sample

 The patient consent form (Form G) and TRF for prenatal cases must be duly filled with the
PCPNDT registration number and stamp of the clinician and seal of the hospital/clinic where
procedure has been performed. These are requirements from the PCPNDT which all genetic
laboratories have to adhere by

 For all prenatal diagnostic samples sent for molecular genetic testing, parental blood samples
in EDTA are mandatory in order to rule out maternal cell contamination.

 Acceptable Specimen for PoC:Placental tissue, Fetal cord blood, Soft tissue ( Ear pinna, Butt
skin, Tip of Finger

 Unacceptable Specimen for PoC: Whole Fetus, Tissues received in unsterile conditions

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  List of few Important People

1. Ujwala Malandkar ( HR ) - 7045695315

2. Nikhil Lonishte (Jr. HR) - 9152096824
3. Hemant Singh ( Sample Pick up )- 7506335967
4. Shivanjali Kapse ( Genetic counseling) - 9152096827
5. Pooja Rayasam ( Genetic counseling) - 9176840878
6. Rohit Asrani ( Accounts & Finance ) - 9867118456
7. Vasudha Shethia ( Administration ) - 9920603366
8. Rakesh Pandya ( GM Sales ) - 7045695166
9. Chaitra Parulekar ( Medical Science Liason ) - 7506335968
10. Lilac Insights Reception- 02241841438, 02241841404
11. Milap Sawant ( Training Manager ) - 7045458614
12. Jenil Ashani ( Asst. Training Manager) - 7506014009
13. Abhijeet Goon (GM- Marketing)- 9920445580

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