Rapid aneuploidy screening with fluorescence in-situ hybridisation: is it a sufficiently robust stand-alone test for prenatal diagnosis?

Abstract

To assess the clinical utility of fluorescence in-situ hybridisation with chromosomes 13, 18, 21, X and Y as a stand-alone test in detecting chromosomal abnormalities, and the types of chromosomal abnormalities missed.
Retrospective analysis.
A restructured Government hospital in Singapore and an academic hospital in the United States.
Cytogenetic data of prenatal specimens and results of fluorescence in-situ hybridisation of 5883 patients performed between January 2000 and August 2007 were reviewed.
Fluorescence in-situ hybridisation detected 558 (9.5%) patients with chromosomal abnormalities. Abnormal ultrasounds (70%) and maternal serum screens (21%) were the most indicative of chromosomal abnormalities. When comparing fluorescence in-situ hybridisation data with karyotype results for the five chromosomes of interest, the sensitivity and specificity were 99.3% and 99.9%, respectively. When comparing fluorescence in-situ hybridisation data with karyotype results for all chromosomes, the sensitivity decreased to 86.8%, whereas the specificity remained at 99.9%. Of 643 cases with karyotype abnormalities, 85 were fluorescence in-situ hybridisation-negative (false negative rate, 13.2%), which included structural rearrangements, chromosome mosaicism, and other trisomies. Despite abnormal ultrasound indications, fluorescence in-situ hybridisation missed 32 cases which included structural rearrangements, mosaicisms, and other trisomies.
This study does not support fluorescence in-situ hybridisation as a stand-alone test. Institutions supporting fluorescence in-situ hybridisation as a stand-alone test must seriously consider the risks of a missed diagnosis.

Full text

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Objectives To assess the clinical utility of fluorescence in-situ hybridisation
with chromosomes 13, 18, 21, X and Y as a stand-alone test
in detecting chromosomal abnormalities, and the types of
chromosomal abnormalities missed.
Design Retrospective analysis.
Setting A restructured Government hospital in Singapore and an
academic hospital in the United States.
Participants Cytogenetic data of prenatal specimens and results of
fluorescence in-situ hybridisation of 5883 patients performed
between January 2000 and August 2007 were reviewed.
Results Fluorescence in-situ hybridisation detected 558 (9.5%) patients
with chromosomal abnormalities. Abnormal ultrasounds (70%)
and maternal serum screens (21%) were the most indicative
of chromosomal abnormalities. When comparing fluorescence
in-situ hybridisation data with karyotype results for the five
chromosomes of interest, the sensitivity and specificity were
99.3% and 99.9%, respectively. When comparing fluorescence
in-situ hybridisation data with karyotype results for all
chromosomes, the sensitivity decreased to 86.8%, whereas
the specificity remained at 99.9%. Of 643 cases with karyotype
abnormalities, 85 were fluorescence in-situ hybridisation–
negative (false negative rate, 13.2%), which included structural
rearrangements, chromosome mosaicism, and other trisomies.
Despite abnormal ultrasound indications, fluorescence in-situ
hybridisation missed 32 cases which included structural
rearrangements, mosaicisms, and other trisomies.
Conclusion This study does not support fluorescence in-situ hybridisation as
a stand-alone test. Institutions supporting fluorescence in-situ
hybridisation as a stand-alone test must seriously consider the
risks of a missed diagnosis.
Rapid aneuploidy screening with fluorescence in-situ
hybridisation: is it a sufficiently robust stand-alone
test for prenatal diagnosis?
O
A
R
R
IGIN
C
A
L
L
ETI
Key words
Aneuploidy; In situ hybridization,
fluorescence; Karyotyping; Prenatal
diagnosis
Hong Kong Med J 2010;16:427-33
Cytogenetics Laboratory, Department of
Pathology, Singapore General Hospital,
Singapore
AST Lim, PhD, MHGSA
TH Lim, MSc, CG(ASCP)
SK Kee, BSc, CG(ASCP)
SL Tien, M MED, FRCPA
Human Genetics Laboratory, University
of Nebraska, Omaha, United States
MM Hess, MS, CG(ASCP)
R Gilbert, BS, CG(ASCP)
TE Hempel, BS, CG(ASCP)
KJ Anderson, BS, CG(ASCP)
DH Zaleski, BS, CG(ASCP)
WG Sanger, PhD, FACMG
Temasek Applied Science School,
Temasek Polytechnic, Singapore
YYF Lau, DipBio
Fetal Medicine and Gynaecology Centre,
Kuala Lumpur, Malaysia
P Chia, MB, BS, MRCOG
R Subramaniam, MD, FRCOG
Prenatal Diagnostic Centre, Obstetrics
and Gynaecology Department, Singapore
General Hospital, Singapore
HK Tan, M MED, FRCOG
Women and Fetal Centre, Singapore
ASA Tan, M MED, MRCOG
Correspondence to: Adjunct Assoc Prof
AST Lim
Email: alvin.lim.s.t@sgh.com.sg
Introduction
Cytogenetic analysis of fetal cells has been the standard test in prenatal diagnosis. This
method involves the acquisition of metaphase chromosomes through a period of cell
culture that may take anywhere between 7 and 14 days. Despite increasingly shorter
prenatal result turn-around times due to the wider adoption of the in-situ coverslip
technique and improved culture media, patients remain anxious while waiting for the 1
to 2 weeks it takes for an amniotic fluid (AF) or chorionic villus sampling (CVS) karyotype
result.

hybridisation (FISH) and quantitative fluorescence –polymerase chain reaction (QF-PCR)
assays to detect numerical abnormalities of chromosomes 13, 18, 21, X and Y have become
increasingly popular adjunct tests.1,2 Unlike karyotyping, these test results are typically
available within a few hours to 2 days, thereby alleviating much of the anxiety from these
patients.3,4 Since aneuploidies of chromosomes 13, 18, 21, X and Y account for 60 to 80%
of the chromosomal aberrations at the time of prenatal diagnosis,5,6 the prenatal RAS FISH
assay is centred on just these five chromosomes. Commercially available probes for these
five chromosomes have a reported sensitivity and specificity of around 100%.7-10 These
probes have been shown to be highly accurate by several groups.11,12
Consequently, rapid aneuploidy screen (RAS) tests such as fluorescence in-situ
Alvin ST Lim
TH Lim
Michelle M Hess
SK Kee
Yvonne YF Lau
Rebecca Gilbert
Thomas E Hempel
Kirby J Anderson
Dianna H Zaleski
SL Tien
Patrick Chia
Raman Subramaniam
HK Tan
Ann SA Tan
Warren G Sanger

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?目的? 評估染色體13、18、21、X和Y熒光原位雜交術作為
染色體異常獨立測試的臨床意義，以及分析漏診的染
色體異常類別。
?設計? 回顧分析。
? 安排? 新加坡一所重建政府醫院和美國一所學術醫院。
?參與者? 回顧2000年1月至2007年8月期間，5883名病人的產
前檢查數據和熒光原位雜交術結果。
? 結果? 熒光原位雜交術能檢測到558名（9.5%）病人呈現染
色體異常。異常的超聲波（70%）和孕婦血清檢查結
果（21%）最有效顯示染色體異常。把上述五種染色
體的熒光原位雜交術數據與染色體核型結果比較時，
敏感性和特異性分別為99.3%和99.9%。把所有染色體
的熒光原位雜交術數據與染色體核型結果比較時，敏
感性減至86.8%，特異性則維持99.9%。在643宗染色
體核型異常病例中，85宗的熒光原位雜交術結果呈陰
性（錯誤負判率，13.2%），這包括基因結構重組、
鑲嵌型染色體和其他三體綜合徵。儘管超聲波呈異常
結果，但熒光原位雜交術也漏診32宗基因結構重組、
鑲嵌型染色體和其他三體綜合徵病例。
?結論? 上述研究並不支持熒光原位雜交術作為獨立測試。醫
療機構若考慮使用熒光原位雜交術作為獨立測試，必
須慎重考慮有漏診的風險。
以熒光原位雜交術進行快速非整倍體測試：
獨立測試能否提供準確的產前診斷？

RAS as the primary test, replacing chromosome
karyotyping altogether and using it only if indicated
by an abnormal ultrasound (AU).13-15 In 2004, the
UK National Screening
recommended new screening programmes for
Down syndrome with FISH or QF-PCR, that could be
offered as a rapid stand-alone diagnostic test instead
of karyotyping. The UKNSC also suggested that it
should be offered as part of the UK National Health
Service provision to all women undergoing invasive
testing.16
Of late, calls have been made to introduce
Committee (UKNSC)

RAS have been made on the basis of purported cost-
savings for patients and more efficient utilisation of
limited clinical resources. Because it is expensive
to perform RAS and karyotyping on all prenatal
samples, various strategies involving RAS screening
have been proposed.17,18 One option is to offer FISH
for chromosomes 13, 18, 21, X and Y as a stand-alone
test to patients without an AU finding but with a
clinical indication, namely advanced maternal age
(AMA) or abnormal maternal serum screening (MSS).
Karyotyping following FISH would be reserved only
for those with an indication based on AU.
These proposals to replace karyotyping with

of the assays, aneuploidy detection by FISH and
Despite the very high sensitivity and specificity
QF-PCR have their own serious limitations. These
include their innate inability to detect unbalanced
structural rearrangements, trisomies other than
chromosomes 13, 18, 21, X and Y, and numerical
mosaicism that can result in birth defects. For this
reason, RAS is usually performed in many laboratories
as an adjunct to karyotyping.

utility of the FISH assay with probes for chromosomes
13, 18, 21, X and Y as a stand-alone test in detecting
chromosomal abnormalities, and the types and
frequencies of chromosomal abnormalities missed.
To evaluate these, archival reports of cases that
had both karyotype and interphase FISH tests were
reviewed and analysed retrospectively.
The aim of this study was to assess the clinical
Methods
Patients
The cytogenetic data of AF and CVS and interphase
FISH analyses of patients performed between
January 2000 and August 2007 at the Cytogenetics
Laboratory (Singapore General Hospital, Singapore)
and the Human Genetics Laboratory (University of
Nebraska Medical Center, Omaha, US) were retrieved
and reviewed. A total of 5883 patient samples
were included in this analysis. Amniocentesis was
generally performed at around 16 weeks of gestation
and usually 20 mL of fluid was collected. Chorionic
villus sampling was performed at round 12 weeks of
gestation and usually 15 mg were obtained.
Fluorescence in-situ hybridisation
Fluorescence in-situ hybridisation probes
The FISH assay employed the AneuVysion Assay
Kit (Abbott Molecular, US) comprising CEP 18
(SpectrumAqua), CEP X (SpectrumGreen), CEP Y
(SpectrumOrange), LSI 13 (SpectrumGreen), and LSI
21 (SpectrumOrange) probes.
Amniotic fluid
About 2 to 5 mL of uncultured AF samples were used
for each assay. Amniotic fluid samples were prepared
according to the protocol recommended by the
manufacturer.
Chorionic villus
The villi were cleaned under a stereomicroscope to
ensure no maternal decidua remained before they
were processed. Approximately 5 mg of villi were
processed for each FISH assay. The chorionic
villi were prepared according to the protocol
recommended by the manufacturer. The slides were
labelled with the lab number, patient’s initials, and
FISH probes to be used.

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Fluorescence in-situ hybridisation protocol
Probe mixtures were added onto the target areas of
the slides, coverslipped and the edges sealed with
rubber cement. Co-denaturation and hybridisation
were carried out in a HYBrite system (Abbott). The
next day, the slides were washed and prepared for
analysis. Fifty nuclei were scored for each probe.
Results
From January 2000 to August 2007, both centres
collectively processed a total of 5883 cases with both
karyotyping and FISH requests.

included AMA (≥35 years) [24.2%], AU findings
(33.6%), abnormal MSS (34.9%), family history of a
genetic or chromosomal disorder (5.8%), parental
anxiety (0.6%), and others such as in-vitro fertilisation
pregnancy. In cases where there were multiple
indications, priority of indication was assigned as
follows: AU, MSS, and AMA. About a third of each
of all indications were for AU findings and abnormal
MSS (Table 1). Conventional karyotyping was carried
The clinical indications for prenatal studies
out concurrently for all cases. A minimum of 15
colonies or 20 cells were analysed for each case.
To exclude mosaicism, two or more cultures were
analysed per case.

abnormalities were detected by FISH (Table 2).
Trisomy 21, as expected, was the most frequent
chromosomal disorder across all indications (42.1%).
Some of the other abnormalities were trisomy 18
(25.3%), monosomy X (14.0%), trisomy 13 (12.4%),
triploidy (3.6%), and other sex chromosome
disorders (2.2%), in descending order. The majority
of the abnormalities identified by FISH were due to
AU (70.1%) and MSS (21.0%); AMA constituted 6.6%
of the abnormal cases while family history and other
indications accounted for the remainder. There were
no abnormalities detected for parental anxiety.
A total of 558 (9.5%) cases of chromosomal

karyotype results for chromosomes 13, 18, 21, X
and Y in the 5883 individuals tested, there was only
one false-positive result, but there were four false-
negative results, giving a sensitivity of 99.3% and a
specificity of 99.9% (Table 3). The false-positive rate
was 0.02% while the false-negative rate was 0.7%.
The sole false-positive case was due to a balanced
translocation between chromosomes 9 and 18 at
bands p11.2 and p11.1, respectively. This led to a split
chromosome 18 centromere signal resulting in three
interphase FISH signals. The false-negative results
were due to samples that were heavily bloodstained
compounded by technical difficulties.
When comparing the FISH data with the

detect aneuploidies of only the five chromosomes,
it follows that there would be abnormalities that are
undetectable by FISH when the FISH performance
is compared with the karyotype results for all
As the FISH assay is specific and designed to
IndicationNo. (%) of samples
(n=5883)
Advanced maternal age (≥35 years) 1422 (24.2)
Abnormal ultrasound 1974 (33.6)
Abnormal maternal serum
screening
2054 (34.9)
Family history of genetic/
chromosomal disorder
340 (5.8)
Parental anxiety 35 (0.6)
Others 58 (1.0)
TABLE 1. Clinical indications for prenatal studies
Indication Trisomy
13
Trisomy
18
Trisomy
21
Mono-
somy X
XXYXXX XYYTriploidyMosai-
cism
Total No.
(%) of
abnormal
cases
Advanced
maternal age
(≥35 years)
08 26111000
37 (6.6)
Abnormal
ultrasound
62 113 13365200151
391 (70.1)
Abnormal
maternal serum
screening
6 19 711201242
117 (21.0)
Family history of
genetic/
chromosomal
disorder
003000000
3 (0.5)
Parental anxiety000000000
0 (0)
Others112050010
10 (1.8)
Total69 (12.4) 141 (25.3)235 (42.1)78 (14.0) 8 (1.4)2 (0.4)2 (0.4)20 (3.6) 3 (0.5)558
TABLE 2. Detection of abnormal cases by fluorescence in-situ hybridisation according to various indications

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chromosomes. Indeed, the sensitivity dropped to
86.8% while the specificity remained at 99.9% (Table
4). Of 643 cases with an abnormal karyotype, there
were 85 false-negative cases by FISH. The false-
negative rate was 13.2%.

rearrangements,
and trisomies of chromosomes other than the
aforementioned (Table 5). While the FISH pick-up
rate was 9.5%, the total abnormality rate by
karyotyping was 10.9% (643/5883). Among cases that
had various indications, including AU, there were
85 cases that were missed by FISH. These included
balanced structural rearrangements (30 cases),
unbalanced structural rearrangements (34 cases),
mosaicism of chromosomes 13, 18, 21 and the sex
chromosomes including mosaicism of structurally
rearranged chromosomes (13 cases), and trisomies
of chromosomes other than the five included in the
FISH assay (8 cases). With AU alone as the indicator,
The FISH-negative cases included structural
chromosome mosaicism,
there were 32 cases that were missed by FISH.
Discussion
Conventional
chorionic villus tissues detects both chromosome
aneuploidies and structural rearrangements with
up to 99.5% accuracy.19,20 Owing to the need for cell
culture however, a test may take anywhere between
1 and 2 weeks before it is completed due to the
long culture time. Such long waiting period places
significant emotional and psychological stress on
patients and families.21,22 The introduction of RAS to
prenatal diagnosis has alleviated many of problems
associated with the long wait. Because aneuploidies
of chromosomes 13, 18, 21, X and Y are the most
frequent, the prenatal RAS FISH assay is centred on
just these five chromosomes.
cytogenetic analysis of AF or

chromosomes 13, 18, 21, X and Y detected 558
In this study, FISH using probes for
Abnormal karyotype
(for 13, 18, 21, X and Y)
Normal karyotype
(for 13, 18, 21, X and Y)
Total
Abnormal FISH (TP†) 558(FP) 1§
559
Normal FISH (FN) 4¶
(TN‡) 5320
5324
Total 5625321 5883
TABLE 3. Abnormalities detected by fluorescence in-situ hybridisation (FISH) versus karyotyping for chromosomes 13, 18, 21, X and Y*
* TP denotes true positive, TN true negative, FP false positive, and FN false negative
† Sensitivity (true positive rate)=558/562=99.3%
‡ Specificity (true negative rate)=5320/5321=99.9%
§ One false-positive case due to fetus with a balanced t(9;18)(p11.2;p11.1) resulting in a split 18 centromere
¶ Four false negatives due to suboptimal samples and technical problems
Abnormal karyotype Normal karyotypeTotal
Abnormal FISH (TP†) 558(FP) 1
559
Normal FISH (FN) 85(TN‡) 5239
5324
Total6435240 5883
TABLE 4. Abnormalities detected by interphase fluorescence in-situ hybridisation (FISH) versus karyotyping for all chromosomes*
* TP denotes true positive, TN true negative, FP false positive, and FN false negative
†
Sensitivity (true positive rate)=558/643=86.8%
‡
Specificity (true negative rate)=5239/5240=99.9%
Indication*Balanced
structural
karyotype
Unbalanced
structural
karyotype
MosaicismTrisomies other
than 13, 18, 21,
X and Y
Total
Advanced maternal age (≥35 years) 14452
25
Abnormal ultrasound6 1556
32
Family history of genetic/chromosomal disorder614 00
20
Abnormal maternal serum screening4120
7
Parental anxiety0010
1
Other indications0000
0
Total 3034 138
85
TABLE 5. Chromosomal abnormalities by various indications that were normal by fluorescence in-situ hybridisation
* Excluding abnormal ultrasound indications, 8 cases had termination of pregnancy, 1 case ended in a miscarriage, 7 cases with malformations, 11 normal births,
and 2 cases untraceable

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abnormalities (Table 2). Trisomies 21, 18, monosomy X
and trisomy 13 were the most frequent abnormalities
in descending order. Among all the indications, an
AU and MSS are the most critical in determining the
requirement of a prenatal diagnosis.

assay versus karyotyping results were calculated to
be 99.3% and 99.9%, respectively (Table 3). Of the
5883 cases, there were 558 true-positive cases, four
false-negative cases and one false-positive case. The
four false-negative results were due to technical
problems one of the laboratories was facing at the
time as well as poor sample quality. The single false-
positive was due to a fetus with a balanced t(9;18)
(p11.2;p11.1) rearrangement that resulted in a split
chromosome 18 centromere, giving rise to three
copies of chromosome 18 signals. One parent was
found to carry the same rearrangement. The baby was
born clinically normal. The results of the FISH assay
are comparable to those reported in the literature
and attest to the high specificity and sensitivity of
the FISH assay in the detection of aneuploidies of
chromosome 13, 18, 21, X and Y when compared
with karyotyping, which is long considered the gold
standard method of detection. When the detection
rate of the RAS FISH was compared with karyotyping
for all chromosome abnormalities, the sensitivity and
specificity values were 86.8% and 99.9%, respectively
(Table 4). Thus, when all chromosomal abnormalities
were considered as opposed to just the five that were
targeted, the specificity remained constant at almost
100% but the sensitivity had dropped by 12.5%. This
difference was due to 85 false negatives (missed by
FISH but detected by karyotyping). Among these, AU
missed a total of 32 cases (Table 5). Excluding balanced
structural rearrangements, there were a total of 55
unbalanced karyotypes with potential phenotypic
abnormalities. Of these, an AU indication incurred
the highest frequency with 26 (47%) cases compared
with the other indications. Notwithstanding this
finding, there were 29 abnormal cases in which there
was a normal ultrasound. In these chromosomally
unbalanced cases, abnormalities were detected
only after birth or termination of pregnancy (TOP).
Of these, family history of genetic/chromosomal
disorder accounted for the highest number of cases
(14), followed by AMA (11), MSS (3), and parental
anxiety (1). This indicates that not only AU and
MSS (Table 2) are important indicators of prenatal
diagnosis, AMA and family history are also important.
In the context of a normal ultrasound finding and
MSS, offering RAS FISH as the sole genetic test with
indications of AMA or FH will lead to underdiagnosis
in certain circumstances.
The sensitivity and specificity of the RAS FISH

normal ultrasound findings were likely to have been
viable had the pregnancies been sustained. There
were eight that underwent a TOP and one ended in
Some of the above-mentioned 29 cases with
a miscarriage. Two were lost to follow-up while the
remainder went to term. Of those that went to term,
seven were born with a wide range of malformations
while the others were born clinically normal. Of
the TOP cases and those born with malformations,
the histopathological reports and neonatological
findings of multiple congenital abnormalities were
consistent with the features expected with the
specific chromosomal abnormality. The chromosomal
abnormalities of the TOP cases included imbalances
due to deletions, partial trisomies due to adjacent-1
and 3:1 segregation
translocations, two cases of familial duplication of
3q syndrome due to insertions, and mosaicism for
trisomies 12 and 14.
patterns of reciprocal

this study was 10.9% (643/5883 cases) of which FISH
detected over 91% (588/643). This figure is similar to
the 11% abnormality rate reported by Dickinson et
al,22 but much higher than the 4 to 4.8% rate obtained
by Dupont and Carles23 and Leclercq et al.17 This
apparent discrepancy may be due to our patient
profile that more closely matched Dickinson’s
patients22 in having a higher proportion assessed for
MSS and AU and fewer for AMA,17 thereby resulting
in a higher abnormality rate.
The total abnormality rate by karyotyping in

a normal result is obtained by the RAS FISH assay,
which is of primary importance. However, the final
and conclusive result is not available until a full
karyotype is obtained in the week following the
FISH findings, in which abnormalities not detected
by FISH can be unveiled. Indeed, evidence has been
mounting that RAS is unable to replace conventional
karyotyping owing to the number of misdiagnoses in
the absence of ultrasound abnormalities.24,25 Several
very large retrospective studies were carried out to
address this issue. Thus, Caine et al6 looked at some
119 528 AF samples and 23 077 chorionic villus samples
and showed that stand-alone FISH missed around
1% of all prenatal chromosomal abnormalities, of
which about a third might carried a significant risk of
serious phenotypic consequences. Evans et al5 led an
international multicentre collaborative assessment
of 146 128 prenatal samples and determined that
approximately 0.9% of abnormal karyotypes would be
missed if only FISH were employed, notwithstanding
the contribution of AU was not factored into the
pick-up rates.
There is much reassurance for parents when

strategy used by proponents of RAS FISH as a stand-
alone test, and to use karyotyping only if there
is an ultrasound abnormality, is unreliable. Many
abnormalities will be missed. In this study, although
the 18 cases with malformations (Table 5 footnote,
including the 2 cases lost to follow-up) comprised
only 0.3% of all the cases investigated by FISH and
The evidence from this study shows that the