Pranita Pai, Anju Shukla and Girisha K M Department of Medical Genetics, Kasturba Medical College, Manipal University, Manipal - 576 104 Email:pranitapai@gmail.com
1 Introduction
Chromosomal abnormalities, both numerical and structural, are known to occur in approximately 1 in 200 live births. The
diagnosis for chromosomal abnormalities in the antenatal period is usually done by conducting cytogenetic analysis of
amniotic, chorionic or fetal blood cells obtained by invasive procedures. Karyotyping is a well-established
cytogenetic technique which has been extensively used as a diagnostic tool for pregnant women undergoing these
procedures. The technique is considered 100% sensitive and specific and the gold standard for the detection of
autosomal trisomies and sex chromosome aneuploidies, against which all other techniques are compared. In
addition to chromosomal aneuploidies, structural rearrangements and triploidy can also be detected with a
resolution of 10 million DNA base pairs. The various indications for fetal cytogenetic testing include: 1)
abnormal ultrasound scan, 2) abnormal maternal serum biochemical results, 3) advanced maternal age (≥35
years at the expected time of confinement) 4) family history of chromosomal aberrations or other genetic
disorders.
The rapid rise in the number of pregnancies undergoing maternal biochemical screening and targeted anomaly scans
has led to increase in the number of invasive prenatal diagnostic procedures as well. Rapid diagnosis of aneuploidies in
these cases is warranted for appropriate management of pregnancy as well as to relieve anxiety for the family. The legal
limit of 20 weeks for pregnancy termination in India is a unique legal circumstance which calls for early detection of these
abnormalities.
The autosomal trisomies constitute 80% of the chromosomal aberrations. Hence, Rapid Aneuploidy Detection (RAD)
methods, which are targeted methods for the diagnosis of common autosomal trisomies (13, 18, 21) and sex
chromosome aneuploidies are offered in cases of invasive prenatal testing. Three methods i.e. Fluorescent In Situ
Hybridization (FISH), Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR), and Multiplex Ligation
Dependent Probe Amplification (MLPA) have been validated for use in prenatal diagnosis (Mann et al.,
2004).
∙Fluorescent In Situ Hybridization (FISH) technique: It is usually performed on uncultured interphase cells
with probes designed specifically for chromosome 13, 18, 21, X and Y. The number of fluorescent signals per cell gives the
number of copies of the targeted chromosome (Mann et al., 2004). The technique is known to be almost 100% sensitive
and specific for detection of aneuploidies (Grimshaw et al., 2003). Another advantage is the capacity to
detect triploidy where an extra set of chromosomes is present in the cell. However, the FISH technique is
non-automated, time consuming and necessitates a skilled technician Mann et al., 2004; Grimshaw et al.,
2003).
∙Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR): This assay has been widely used for
the past 20 years for rapid aneuploidy detection. It is a PCR based molecular method which uses fluorescent labeled
primers to amplify specific DNA markers which are polymorphic (STRs) and unique for chromosomes 13, 18, 21,
X and Y. The amplified products are separated through capillary electrophoresis. The copy number of a
specific sequence of each chromosome is determined based on the intensity of the fluorescent signal. The
sensitivity and specificity of the assay is in the range of 95% -100% (Grimshaw et al., 2003; Cirigliano et al.,
2009). There are several commercially available QF-PCR kits (Aneufast TM, Chromoquant aneuploidy
detection kit) (Allingham-Hawkins et al., 2011). Detection of maternal cell contamination, triploidy and
mosaicism as low as 15% are important advantages of these techniques (Mann et al., 2004; Cirigliano et al.,
2009). However, extensive blood staining of specimens interferes with the results and interpretation of this
test.
∙Multiplex Ligation Dependent Probe Amplification (MLPA): It is also a PCR based method. It is
relatively cheaper and less labor intensive than the FISH technique. The technique involves the use of two fluorescent
labeled probes which are hybridized to specific DNA sequences and are then joined by the enzyme DNA ligase. The free
ends of the ligated probes are complementary to the primer which enables the amplification of target sites. The amplified
products are separated based on size using capillary electrophoresis. Each peak is considered to be the amplified product
of a specific probe. The technique has a capacity to quantify up to 40-50 different target sequences in one
reaction. The commercially available kit MLPA P095 kit is useful for the detection of chromosome 13,
18, 21, X and Y chromosome aneuploidies. For detection of aneuploidies, a sensitivity and specificity of
100% is attained by employing this technique (Van Opstal et al., 2009). One of the major drawbacks of
this technique is the failure to detect triploidies especially in a female fetus. It is a completely automated
method, and is being increasingly used as a method for RAD especially where large scale testing of samples is
required.
Additional newer techniques are available such as the chromosomal microarray technique (CMA) which has the
capacity to detect targeted submicroscopic deletions and duplications other than aneuploidies in the prenatal
samples.
2 Non-invasive prenatal testing (NIPT)
In addition to invasive procedures there are non-invasive screening methods which are gaining importance in recent times.
Non-invasive prenatal testing (NIPT) has been widely accepted as a part of routine care for pregnant women in many
countries. The testing involves analysis of cell-free DNA fragments which circulate in the blood of the pregnant women. It
can be offered after 10 weeks of gestation. This technique has a sensitivity of 99% and a specificity of 99.92% for trisomy
21. For trisomy 18, the test has 96.8% sensitivity and 99.85% specificity and for trisomy 13, 92.1% and
99.80% respectively (Gil et al., 2014). The positive predictive value of this technique is approximately 45%
(10 times better than the other maternal biochemical screening tests) for low risk women. The obvious
advantage of this technique is that it provides an alternative for invasive testing and the related complications.
However, currently NIPT is still considered a screening test and the aneuploidy should be confirmed by an
invasive definitive test. It can be used as a first tier test in the first trimester or after abnormal results of the
biochemical screening tests for women unwilling to opt for invasive procedures. However, there are numerous
factors including twin/triplet pregnancies, inadequate fetal fraction of DNA, higher maternal body mass
index, gestational age of less than 10 weeks etc. which can result in either failure or inaccurate results. Also,
the test is unable to detect triploidy in the fetus. To date this testing is available in the US, Europe and
some Asian countries including India. High cost and availability are also limitations for the use of this
technique.
Table 1 summarizes the principles, techniques, advantages and limitations of the important rapid aneuploidy detection
tests. Prior to ordering any of these tests, appropriate pretest counseling is essential.
Description
QF-PCR
FISH
MLPA
NIPT
Principle of
the
technique
Selective
amplification of
genomic DNA regions
(STRs) by binding of
fluorescently labeled
primers to the target
sequences.
The products are then
separated by size
Binding
of a fluorescently
labeled
probe specific for a
DNA sequence and
visualized using a
microscope
Two
probes of unique
length hybridized
to target DNA
sequences
and joined by DNA
ligase. The
amplified target
sites separated by
size
Sequencing of cell free
fetal DNA present in the
maternal plasma
False result
findings
No false-positive,
minimal false
negative results
False-positive rate
of less than 1 in
30,000 cases and
false-negative rate
of
less than 1 in 4000
(Tepperberg et al.,
2001)
–
False-positive rates
0.1%-0.2% (Bianchi et
al., 2014)
Sensitivity
98.9%*
(Allingham-Hawkins
et al., 2011)
95.65% (Cirigliano et
al., 2009)
100%
100%
99% for trisomy 21,
96.8% for trisomy 18,
92.1% for trisomy 13
(Gil et al., 2014)
Specificity
100%*
(Allingham-Hawkins
et al., 2011)
99.97% (Cirigliano et
al., 2009)
100%
100%
99.92% for trisomy 21,
99.85% for trisomy 18,
99.80% for trisomy 13
(Gil et al., 2014)
Mosaicism
Can detect as low as
15% (Mann et al.,
2004)
Standard
practice is to score
100 cells to exclude
mosaicism at a level
of greater than 10%
to 15%, a level
similar to that of
full karyotype
Unknown
sensitivity for
mosaicism. Further
detection has to be
done by employing
the FISH technique
Interpretation can be
altered by presence of
mosaicism
Maternal cell
contamination
Results
cannot be obtained in
heavily blood stained
samples (1%) due to
the presence
of MCC (Cirigliano et
al., 2009)
MCC
can rarely interfere
with interpretation.
Although it is less
sensitive to MCC,
in
female fetus MCC
goes undetectable
Female
fetus detection of
MCC not possible.
In male fetuses the
evidence of MCC is
examined from the
results of
probes located on X
chromosome
–
Cost
Due to automated
methods, this
technique is relatively
cheaper than the
FISH method.
However commercial
kits can increase the
cost of diagnosis per
sample
Comparatively
more expensive
than QF-PCR
Cost comparable to
QF-PCR
Current cost is high
Turnaround
Time
Average is 30.5 hours,
Median is 25.1 hours
(Allingham - Hawkins
et al., 2011)
Reported within
24-72 hours
30 hours (Van
Opstal et al., 2009)
Mean turnaround time
is 5.1 business days
(Bianchi et al., 2014)
Advantages
Reliable, automated,
detects triploidy
and mosaicism. MCC
problems minimized
Reliable for
detection of
targeted
aneuploidies.
Detects
triploidy and MCC
does not interfere
with interpretation
Low cost and
amplification
of different markers
in one tube
Rapid method and
accurate for detection of
Down syndrome
Disadvantages
Commercial
kits may increase the
cost. Cannot detect
structural
chromosomal
aberrations
Non-automated,
requires skilled
technician, labour
intensive, intact
cells, considerable
time, fails to detect
balanced
rearrangements
and imbalanced
aberrations of
chromosomal
segments
Cannot detect all
cases of triploidy,
and sensitivity to
mosaicism is
unknown.
MCC detection not
possible in female
fetus. Cannot
detect structural
chromosomal
aberrations
Need to confirm
aberrant findings
using invasive methods.
Relatively
high percentages of test
failures rates (approx
5%) (Bianchi et al.,
2014)
Kits used
Aneufast TM and
Chromoquant
(Cirigliano et
al., 2009; Allingham -
Hawkins et al., 2011)
Table 1: Comparison of different techniques used for rapid aneuploidy detection.
3 Conclusion
There are different rapid prenatal tests and options which can be offered to pregnant women. However, even with different
options available, a rapid prenatal aneuploidy test should meet certain important criteria: (1) vastly accurate with less
number of false-negative results; (2) no false-positive results because certain important irreversible decisions such as
pregnancy termination may be taken as a result of an abnormal result; (3) robust with minimum failure rates and
ambiguous results; (4) rapid with high specimen throughput; (5) cost effective, as the rapid test is likely to be
conducted in addition to full karyotype analysis; (6) efficient in detection in specimens of low quality and
quantity and (7) sensitive to detect MCC and mosaicism (Mann et al., 2004). Counseling, both pre and
post test, should accompany the above mentioned testing to facilitate informed decision-making for the
family.
References
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