TRISOMY UPDATE

REPORT

Current trends in
antenatal screening
for Downs syndrome
A report from the recent Roche Antenatal Screening Educational Day
by independent healthcare correspondent Sue Libretto.

MARCH 2011

PRENATAL SCREENING
The birth prevalence of Downs syndrome
increases with maternal age. The risk of
having children with the disorder is one in
1250 for women under the age of 24,
increasing to about one in 14 at the age
of 48.3 This trend is compounded by changes
in family planning over time, with women
now opting to have children later in life
(Fig 1). A maternal age of 35 or older at the
time of delivery has been used historically to
identify women at highest risk of carrying a
child with Downs syndrome.
Diagnostic prenatal testing, such as
amniocentesis and chorionic villus sampling
(CVS), is used to obtain tissue or cells of

SCREENING STRATEGIES
Dr Hofmann described the current screening
strategies for Downs syndrome, where the
challenge is to obtain a high detection rate
(DR) with a low false-positive rate (FPR).
Figure 2 shows the main two methods of
screening for Downs syndrome using
ultrasound and serum markers. Nuchal
translucency (NT) is a first-trimester
ultrasound marker monitored between the

600

Live birth per 1000 females

Downs syndrome, also known as trisomy 21, is

a common chromosomal anomaly that results
from an extra copy of some or all of the genetic
material on chromosome 21.1 The disorder is
associated with a set of distinctive physical
features, a number of major structural
congenital malformations (eg heart and
gastrointestinal defects) and mental
retardation. Affected individuals also have
an increased risk of developing leukaemia,
thyroid disorders and Alzheimers disease.
Downs syndrome can have a major impact
on a family. Screening tests have been
available for over 30 years to enable timely
medical or surgical treatment before or after
birth, and to give parents the opportunity to
prepare for a disabled child or to abort the
fetus with the diagnosed condition. Driven by
the National Institute for Health and Clinical
Evidence (NICE) guidance, the NHS
recommends screening to be completed by
the end of the first trimester.2
With the aim of providing an update on
the current trends in antenatal screening for
Downs syndrome, Roche hosted an
educational seminar last November for
laboratory professionals. Chaired by Ian
Parfrement, sales and marketing director of
hospital in vitro diagnostics, Roche UK, the
seminar also heard from Dr Verena Hofmann
from Roche, Professor Kevin Spencer,
Director of Biochemical Screening at the
Fetal Medicine Foundation, Wayne Huttly
from the Wolfson Institute, Barts and The
London School of Medicine, and Alan James,
a biomedical scientist at the Royal Devon and
Exeter Hospital Trust, all of whom have
considerable expertise in antenatal screening
for Downs syndrome.

placental or fetal origin, without removing

any tissue from the fetus itself. Their invasive
nature remains a major concern due to
procedure-induced loss of the fetus.
To reduce the number of invasive
procedures, screening methods using multiple
markers have emerged over the past 20 years,
achieving wide acceptance in routine
antenatal care. These screening tests are used
to identify women with an increased risk of
a Downs syndrome fetus.

500

1980
2000

400
300
200
100
0
<20

2024

2529

3034

3539

4044

45

Mothers age at birth

Fig 1. Age-specific birth rates in Germany 1980 and 2000 showing trend towards older
maternal age at child birth (Source: Council of Europe [www.coe.int]).

THE BIOMEDICAL SCIENTIST

175

Screening Strategies for Down Syndrome

Risk assessment in first and second trimester

REPORT
11th and 13th week of gestation to measure
the amount of fluid accumulated around the
back of the neck of the fetus. Serum markers
are used in either the first or second
trimester, at 1114 weeks or at 1520 weeks,
respectively. There are six commonly used
serum markers, the median concentration
of each varying with gestational age. For this
reason, the concentration of each marker is
expressed as a multiple of median (MoM) for
unaffected pregnancies of a given gestational
age.
While the integrated screen comprising
combinations of measurements from the
first and second trimesters leads to higher
DRs and lower FPRs (about 8095% for
a 5% FPR) than other tests, Dr Hofmann
demonstrated that first-trimester screening is
highly effective, achieving detection rates of
7889% for a 5% FPR.

FIRST-TRIMESTER
SCREENING MARKERS
Pregnancy-associated plasma protein A
(PAPP-A) and free -human chorionic
gonadotrophin (free hCG) are the two
current first-trimester screening markers,
explained Dr Hofmann. PAPP-A is a
metalloproteinase secreted by the placental
trophoblast and released into maternal
circulation. It is detectable at about six weeks
of pregnancy, and increases steadily until
term. PAPP-A is decreased by about 50%
of the normal median value in affected
pregnancies. The marker has discriminatory
power in the first trimester only, as PAPP-A
levels in the second trimester are equal to
those in unaffected pregnancies (MoM 1.0).
Human chorionic gonadotrophin, a
glycoprotein hormone produced by the
syncytiotrophoblast and secreted into the
bloodstream, is active only as a holohormone
consisting of an and subunit. The assay
utilises the free subunit of hCG, which
comprises only a minor fraction of total hCG
(~1%) in the blood. Its concentration rises
exponentially in the first trimester, reaching
a maximum at approximately week 10 of
gestation. Concentrations then decrease to
approximately one-fifth of the maximum
level until week 16 and remains at this level
until term. Free hCG is increased by about
100% of normal median values in affected
pregnancies.

SCREENING PROGRAMMES
AND STANDARDS
Screening using multiple markers has been
available to women in the UK for over 20
years. Today, many tests exist because of
the absence of a coordinated approach and
safety-efficacy focus to screening programme
development. Accordingly, all tests are not
equal in terms of DRs and FPRs, noted
Wayne Huttly. The three most frequently
used tests are the quadruple, combined and
integrated tests. With a DR of 85%, the
FPRs of these tests are 5.5%, 4.5%, and
0.9%, respectively.

176

THE BIOMEDICAL SCIENTIST

Combined
Combined

7889%
78-89%

1st
1sttrimester
trimester

Double
Double

freehCG,
hCG,
PAPP-A,
Free
PAPP-A,
NT NT
hCG+,
hCG+ AFP
, AFP

Triple
Triple

2nd
2nd trimester
trimester

, AFP,
hCG+ AFP,
hCG+,
uE3uE3

Quadruple
Quadruple

uE3,
Inh Inh
A A
hCG+,
hCG+ AFP,
, AFP,
uE3,

Serum
Serum integrated
integrated

PAPP-A,
quadruple
quadruple
PAPP-A,

1st && 2nd

2nd trimester
1st
Integrated
Integrated

PAPP-A,
quadruple
PAPP-A,
quadruple
NT,NT,
00

10
10

20
20

30
30

40
40

50
50

60
60

70
70

80
80

90
90

100
100

Detection rate percentage for 5% FPR

Detection rate [%] for 5% FPR

Fig 2. Performance of different screening tests in the first and second trimesters.

Although the integrated test provides

estimate DRs with confidence over the long
Internal Use Only
the best DR and FPR, the combined testConfidential:
is
term. Findings from the Wolfson Institute
recommended as the UKs national screening
screening programme 20002010 are similar
test, explained Wayne Huttly. The National
to published data, with a DR of 87%
Screening Committee (NSC) established a
(95% confidence interval [CI] 8390%).
National Antenatal Screening Framework in
SAMPLE ANALYSIS AND
2000, and subsequently the Fetal Anomaly
CLINICAL PERFORMANCE
Screening Programme. The original model of
Pregnancy-associated plasma protein A and
best practice recommended the use of tests
free hCG comprise first-trimester screening
that achieved a minimum performance level
markers for the Roche Elecsys 2010 system,
of a 75% DR for a 3% FPR. This was revised
explained Dr Hofmann. The Elecsys PAPP-A
in 2008 to recommend a performance target
and free hCG assays show excellent assay
of more than 90% DR for a FPR of less
performance, giving reliable test results.
than 2% by April 2010. This is currently
Dr Hofmann demonstrated high levels of
unachievable using the combined test, and it
diagnostic precision, repeatability, analytical
is likely that the next model of best practice
sensitivity and specificity using these assays
will suggest it as an inspirational rather
and system. The precision of both assays was
than a real target. The NSC advised that
shown to be good on other Roche analysers,
the combined test should be the preferred
including the cobas e411 and e601 analysers,
method to aid early diagnosis and, at the cost
and were comparable to competitor methods.
of providing the safest and most effective
Dr Hofmann also showed that the Elecsys
screening test, this is supported by NICE.2
free hCG assay correctly recovers the hCG
MONITORING SCREENING
reference material in UK NEQAS ring trial
PERFORMANCE
samples. Recoveries of hCG for the All
All UK laboratories subscribe to external
Method and for single competitor methods
quality assurance schemes, namely the
were too high; as a consequence, the Elecsys
Downs Syndrome Quality Assurance Support
assay data are now included in NEQAS
Service (DQASS) and the National External
reports.
Quality Assurance Scheme (NEQAS).
Clinical performances of Elecsys PAPP-A
Data are submitted biannually to DQASS.
and free hCG assays have been investigated
in a study of 3302 women in weeks 1114 of
Laboratories are provided with a performance
pregnancy, including 32 affected pregnancies.
review of their data compared with expected
Median PAPP-A and free hCG values from
values for various indicators. However,
3270 samples demonstrated that these
DQASS should not be used as an alternative
markers fulfil the Fetal Medicine Foundation
to internal monitoring due to the
requirement of a DR greater than 60% for
retrospective nature of data collection.
a 5% FPR (Table 1).
While it is possible to estimate expected DRs
Alan James shared performance data
based on the age of the women screened,
from evaluation of the Roche free hCG and
individual laboratories can only begin to
Table 1. Risk assessment of Downs syndrome at birth using
Elecsys PAPP-A and free hCG assays.
Risk >1 in 150

Risk at term (without NT)

Risk <1 in 150

Trisomy 21 (n=32)

14 (43.7%)

18 (56.3%)

Unaffected (n=1072)

1018 (97.2%)

29 (2.8%)

MARCH 2011

REPORT
1.5

0.6

a
Identity

0.4

1.0
0.2

Difference*

0.5

Roche

Fig 3. Comparison of free

hCG levels using Roche
and Kryptor methods.
a) Scatter plot log MoM
free-hCG levels;
b) Difference plot MoM
free-hCG levels.

0
0.2

Identity

0.4

0.5

Bias (0.0170222833)
0.6

95% limits of agreement

(0.16773477886 to 0.1336902220)

1.0
0.8

*Roche Log Mom FhCG Kryptor Log Mom FhCG

1.5
1.5

1.0

0.5

0.5

1.0

1.5

1.0
1.5

Kryptor

1.0

0.5

0.5

1.0

1.5

Mean of all

PAPP-A assays for E170 Modular Analytics

carried out at the Royal Devon and Exeter
Hospital. Day-to-day precision of free hCG
and PAPP-A were comparable over 10 days
and to those achieved with the Brahms
Kryptor. There was good correlation between
log MoM free hCG concentrations measured
using the Roche and Kryptor methods (Fig 3).
Similarly, PAPP-A concentrations and log
MoMs of these markers were well correlated
between methods. Precision and quality
control was also demonstrated between kit
lots. The next steps involve comparisons of
median MoMs, risk, and screen-positive rates.

twins. Recent data suggest that use of a

simple correction factor may not be as reliable
as first thought. With vanishing twins, median
MoMs for PAPP-A and free hCG are close to
those for a single pregnancy. If crown-rump
length can be measured (also used to
calculate age at death), high PAPP-A levels
are sustained. Assisted reproduction also
impacts on marker levels.
Having demonstrated in a small study that
PAPP-A levels are lower than normal in women
with insulin-dependent diabetes, Professor
Spencer believes that this, too, will be worthy
of marker-level correction in the future.

Contingency screening raises further

possibilities. After conventional first-trimester
screening, women could be triaged into high,
low and intermediate risk groups to receive
CVS, reassurance, or further screening,
respectively. Ultrasound could be used to
detect additional sonographic markers of
trisomy 21, such as anomalies in the nasal
bone, ductal flow, tricuspid valve or facial
angle. Professor Spencer warned that
ultrasound techniques are difficult and not
for routine screening programmes, but they
may be one way in which second-line testing
could improve DR and FPR.

IMPORTANCE OF MATERNAL
FACTORS IN FIRST-TRIMESTER
SCREENING

FUTURE DEVELOPMENTS IN
FIRST-TRIMESTER SCREENING

VALUABLE INSIGHTS

Screening in the first trimester is about

doing a whole lot more, said Professor
Spencer, who believes that first-trimester
screening is about the detection of fetal
anomalies rather than Downs syndrome only.
In attempting to achieve best practice DRs
and FPRs, Professor Spencer suggested to
take account of maternal factors influencing
serum marker levels. Such factors are
important for risk assessment of the
individual rather than the whole population.
The best estimate of risk is particularly
important for women at borderline risk,
because women tend to modify their
behaviour according to risk level.
The importance of error in dating
gestational age has only recently been
identified. Small errors in crown-rump
length impact on PAPP-A and free hCG, and
therefore on DRs and FPRs. Smoking has a
dose-related effect on PAPP-A, reducing levels
by approximately 20%. Ethnicity is also an
important factor. Weight-corrected median
MoM PAPP-A and free hCG levels are
higher by 55% and 11%, respectively, in AfroCaribbean women compared with Caucasians.
Screening should account for multiple
pregnancies. Pregnancy-associated plasma
protein A and free hCG levels are twice as
high in twin pregnancies than in single
pregnancies, and PAPP-A levels also differ
between monochorionic and dichorionic

178

THE BIOMEDICAL SCIENTIST

There are sound reasons for choosing goodquality assays and systems. However, while
analytical variation is important, it must be
viewed in terms of intra-individual variability.
Professor Spencer believes that future
coefficient of variation targets should be
11.5%, because they significantly reduce
error risk compared to those of 2% or more.
However, this precision is questionable if
biological variability is 10%.
Investigations are focusing on the timing
of serum marker assessment. Early work by
the Fetal Medicine Foundation demonstrating
improved performance of PAPP-A at nine
weeks versus later in the first trimester, and
of free hCG assayed later rather than earlier
in the first trimester, has been confirmed.4
Additional studies have shown that clinical
discrimination of PAPP-A deteriorates before
nine weeks.
Current thinking is to develop different
screening programmes, revealed Professor
Spencer. Options include one appointment at
1113 weeks monitoring NT, free hCG and
PAPP-A, and a first appointment at 912
weeks measuring free hCG and PAPP-A
followed by a second at 12 weeks for NT.
However, a first appointment at 912 weeks
monitoring PAPP-A and a second appointment
at 12 weeks evaluating NT and free hCG
provides the opportunity of using the best
marker for the gestational week, and of
achieving a 90% DR for a 2% FPR.

Concluding the meeting, Ian Parfrement said

that some extremely valuable insights had been
gleaned into the history, current performance,
the foci for future improvements, and
r
developments in antenatal screening.

REFERENCES
1 Mutton D, Alberman E, Hook EB.
Cytogenetic and epidemiological findings
in Downs syndrome, England and Wales
1989 to 1993. National Downs Syndrome
Cytogenetic Register and the Association
of Clinical Cytogeneticists. J Med Genet
1996; 33: 38794.
2 National Institute for Health and
Clinical Excellence. Antenatal care.
Routine care for the healthy pregnant
woman. Nice clinical guidance 62. 2008
(www.nice.org.uk/nicemedia/pdf/
CG062NICEguideline.pdf).
3 Merck. Merck manual of medical
information. Merck, 2003.
4 Kirkegaard I, Petersen OB, Uldbjerg N,
Trring N. Improved performance of firsttrimester screening for trisomy 21 with
the double test taken before a gestational
age of 10 weeks. Prenat Diagn 2008;
28: 83940.
To find out more about Roche meetings
or Downs screening, please telephone
+44 (0)1444 256000 or visit the companys
website (www.roche-diagnostics.co.uk).

MARCH 2011