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CLN’S

LAB 2004:
FROM
BASIC TO
ADVANCED
SERIES
Prenatal Screening for
Down Syndrome
Current Testing and Future Choices
BY JACOB A. CANICK, PHD

S
creening pregnant women for Down syndrome has been a routine part of prenatal care for the
past 10 to 15 years. A survey conducted in 1995 found that about 2.5 million of the 4 million preg-
nant women in the U.S. had screening tests. Today, it is likely that an even higher proportion of
pregnant women undergo screening.
Down syndrome screening currently involves the measurement of multiple biochemical mark-
ers in maternal serum during the early second trimester, between weeks 15 and 20 of a 40-week pregnancy. The
fetus and placenta secrete the markers, and their levels in maternal serum, along with the age of the woman be-
ing screened, indicate the risk of an affected pregnancy. To calculate a risk assessment, the laboratory compares
a woman’s test results to population-based median values and reports them as a multiple of the median (MoM).
The MoM value for each marker contributes to the calculated risk of the woman having an affected pregnancy,
with the combination of MoM values assessed in a statistical algorithm known as multivariate Gaussian distribu-
tion analysis.
Screening Tests: Not a Perfect Tool in prenatal or cancer screening, physicians Down syndrome screening, the challenge
In order to assess the quality of current and offer follow-up diagnostic testing, whereas a is to devise a testing strategy that identifies
emerging prenatal screening tests for Down screen positive individual in cardiovascular the highest number of affected pregnancies
syndrome, it is helpful to review what infor- disease screening would receive treatment. while at the same time selecting the fewest
mation can be obtained from such testing. By and large, most individuals are screen unaffected pregnancies for follow-up.
Medical screening, whether it is prenatal negative and receive routine medical care.
Prenatal Diagnostic Testing:
Reliable, but Risky
Prenatal diagnostic tests can identify fetal
chromosomal abnormalities with almost
perfect accuracy and reliability; however,
such tests are invasive tests and increase the
risk of miscarriage. Amniocentesis is done
beginning at 14 or 15 weeks gestation and is
estimated to cause miscarriage in about 1 in
200 procedures. Chorionic villus sampling
(CVS) is done earlier, between about 10.5
and 14 weeks gestation, but it is riskier than
amniocentesis, with a 1 in 100 risk of mis-
carriage commonly quoted.
Laboratories culture the fetal cells ob-
tained from amniocentesis and placental
tissue from CVS in order to perform the
chromosomal analysis. While the accuracy
of karyotyping is about 99%, the total ex-
pense of the diagnostic procedures is high,
about $1,000. Moreover, if all pregnant
women underwent amniocentesis or CVS,
it is likely that more healthy fetuses would
be lost to miscarriage than those found to be
chromosomally abnormal. Consequently, it
is important to devise testing strategies that
first identify high-risk pregnancies through
screening. Obstetricians can then offer
this smaller group of women the invasive
diagnostic testing in order to definitely de-
screening for serious fetal abnormalities such Two measures determine the perfor- termine whether or not the fetus has Down
as Down syndrome or screening adults for mance of a screening test: the detection rate, syndrome.
cardiovascular disease or cancer, is by defi- or sensitivity, defined as the proportion of
nition imperfect. A screening test can only affected individuals who are screen positive Second Trimester Screening:
assign risk; it can not identify with certainty by the test; and the false positive rate, defined The Current Norm
who is actually affected and who is not. The as the proportion of unaffected individuals In the U.S. and Canada, the most commonly
results of a screening test place an individual who are screen positive by the test. Conse- offered prenatal screening test is the sec-
in either the screen positive or screen negative quently, a good screening test has a high de- ond trimester, triple marker test, or simply
group. When an individual is screen positive tection rate and a low false positive rate. For the triple test. The three maternal serum

12 CLINICAL LABORATORY NEWS AUGUST 2004
Figure 1 Figure 2
Median inhibin A levels (MoM) in Comparison of Down syndrome
Down syndrome pregnancies markers in screening
Down syndrome
Median
Study MoM n Unaffected
Low High
Down syndrome
Wallace ’96 2.60 21
Aitken ’96 2.24 44
Wenstrom ’96 2.20 33
Haddow ’98 2.10 52
Reiner ’98 2.07 32
Lambert-Messerlian ’96 1.95 20
Wald ’96 1.79 77 0.2 0.5 1 2 5 10
NT (MoM)
Yolshida ’00 1.77 15
Spencer ’96 1.77 157 Unaffected
Lam ’99 1.62 49 Down syndrome

Cuckle ’96 1.62 56
D’Antona ’98 1.53 43
All (12 studies) 1.91 599
95% Cl [1.76–2.06] 0.2 0.5 1 2 5 10
hCG (MoM)
0.25 0.5 1 2 3 4

The black circles and bars represent the median (or mean when the median could The figure compares the distributions of nuchal translucency (NT) in the first trimester
not be obtained) and 95% confidence interval and the number of affected pregnan- and human chorionic gonadotropin (hCG) in the second trimester in Down syndrome
cies in each study. Studies are ranked according to the median MoM. The pooled and unaffected pregnancies. Note the much tighter distribution of NT values as com-
median is indicated by the vertical dashed line. pared to hCG values in unaffected pregnancies, making NT a more informative marker
than hCG.
With permission from Prof. N Wald, Wolfson Institute of Preventive Medicine (University of
London, U.K.). Figure courtesy of L. Neveux, Foundation for Blood Research, Scarborough, Maine.

markers measured are alpha-fetoprotein a 70% detection rate has more than doubled commercially in ELISA format (Diagnostic In fact, laboratories in Europe and a
(AFP), which is synthesized by the fetus’ screening performance. Systems Laboratories, Webster, Texas). The small number of programs in the U.S. and
liver; human chorionic gonadotropin assay has FDA clearance as a marker of Canada offer a Down screening test that can
(hCG), which is synthesized by the placenta; Inhibin A as a Second Trimester Marker menstrual cycle function, and in January be done earlier in pregnancy. But is the test
and unconjugated estriol (uE3), which is Over the past eight years, some studies have 2002 clinical measurement of inhibin was better and as readily available and inexpen-
synthesized by the placenta from precursor found a fourth marker, inhibin A, to be use- given its own CPT code (No. 86336). sive as the current second trimester serum
steroids that originate from the fetal adrenal ful in second trimester serum screening for The Foundation for Blood Research screening protocol? The answers to these
gland and liver. While all three analytes are Down syndrome pregnancies. Like the es- (FBR) in Scarborough, Maine, developed questions are not uniformly agreed upon,
used in risk assessment for Down syndrome, tablished screening marker hCG, inhibin A is proficiency testing for prenatal screening and the issues are worth exploring.
it is important to note that AFP is also used a product of the placenta, although it is also in the U.S. 20 years ago. Since 1989, the The first trimester test combines maternal
in screening for open neural tube defects. synthesized in the ovaries. In fact, the name FBR and the College of American Patholo- serum markers and a marker measured in
The performance of the triple test in inhibin comes from its well-established gists (CAP) have jointly administered an an ultrasound examination between 10–11
screening for Down syndrome approaches a hormonal function as a gonadal product external proficiency testing program for and 13 completed weeks of pregnancy. The
detection rate of 70% with a 5% false-posi- that inhibits secretion of the gonadotropin, second trimester serum markers. Two years serum markers currently considered best
tive rate. In other words, the 5% of screened follicle stimulating hormone, at the level of ago, inhibin A was added to the proficiency to use in the first trimester are pregnancy-
women found to have the highest calculated the anterior pituitary gland. Inhibin A is an testing scheme, and according to the latest associated plasma protein-A (PAPP-A) and
risk by the triple test will have 70% of all the alpha-, beta-subunit glycoprotein; the alpha FBR/CAP reports, 60 of approximately 200 the free beta-subunit of hCG. While the
Down syndrome pregnancies. The remain- subunit is common to all forms of inhibin, testing sites that subscribe to the proficiency levels of PAPP-A tend to be low in Down
ing 30% of Down syndrome pregnancies and the betaA subunit is one of at least two testing program have added inhibin A to syndrome pregnancies, the levels of the free
will be missed, falling into the 95% of different beta inhibin gene products. their second trimester serum screening beta-subunit of hCG tend to be elevated.
screened pregnancies that are found to be Based on data from more than 14 pub- protocols. Depending on the gestational age range,
screen negative. lished studies, second trimester levels of hCG and inhibin A are appropriate substi-
In comparison, prior to serum screen- maternal serum inhibin A are, on average, The Option to Screen Earlier in Pregnancy tutes for free beta-hCG. The first trimester
ing, women at the highest risk of a Down about two times higher in Down syndrome Screening for Down syndrome earlier in screen also uses nuchal translucency thick-
syndrome pregnancy were identified solely than in unaffected pregnancies (Figure 1). pregnancy would be desirable, both from ness, or NT, as measured by an ultrasound
on the basis of their age. The rule of thumb Addition of inhibin A to the triple test in- the perspective of patient privacy and, if examination. NT tends to be increased in
was that a woman’s risk increased with her creases the detection rate by 7–10%, giving chosen, safer pregnancy termination. To be Down syndrome pregnancy.
age. According to this method of screening, the so-called “quad marker test” almost an beneficial, an earlier screening test would
the oldest 5% of pregnant women had 30% 80% detection rate with a 5% false-positive have to be as good as what is done in the PAPP-A: A Good Candidate
of all Down syndrome pregnancies, for a rate. second trimester. Moreover, it would need Synthesized and secreted by the placenta,
30% detection rate at a 5% false-positive The immunoassay for inhibin A is a to be easy to perform, readily available, and PAPP-A is a very high molecular weight
rate. In contrast, the current triple test with monoclonal sandwich assay and is available of reasonable cost. tetrameric glycoprotein, made up of two

CLINICAL LABORATORY NEWS AUGUST 2004 13
molecules of pro-major basic protein linked
by disulfide bonds to two unique PAPP-A Figure 3
molecules. PAPP-A concentrations in ma- Performance of various prenatal screening tests for Down syndrome
ternal serum increase throughout preg-
nancy, yet the function of PAPP-A during
pregnancy is poorly understood. Recently,
researchers have shown that it is a protease
25
specific to insulin-like growth factor binding
protein-4, and therefore may be involved in
the regulation of fetoplacental growth.
In Down syndrome pregnancies, mater-
20 21.5%

False Positive Rate
nal serum PAPP-A levels are, on average,
about 2.5 times lower than in unaffected
pregnancies when measured between 8 and
13 gestational weeks, although the extent of 15
the reduction in level is greater earlier in that
time frame. By 14 weeks gestation, PAPP-A 15.2%
loses its effectiveness, and its levels in Down
syndrome pregnancies are similar to those 10
in unaffected pregnancies.
PAPP-A immunoassays are generally 9.9%
configured in a two-site immunometric for- 8.8%
mat. In North America, a number of com-
mercial assays are available, including those
5
from Diagnostic Products Corporation (Los
Angles, Calif.), Diagnostic Systems Labora-
tories (Webster, Texas), and Perkin-Elmer 2.2%
Life Sciences (Norwalk, Conn.). None are 0
FDA-cleared for any purpose and a CPT Triple Quad Combined Serum Full
code has not yet been assigned for clinical Integrated Integrated
measurement of PAPP-A.
Second First Both
NT: A Powerful Marker
trimester trimester trimesters
The NT ultrasound marker differentiates
first trimester screening from second tri- The figure shows the false positive rate needed to achieve a detection rate of 90% . Each screening test combines markers with
mester screening. The measurement is made maternal age in the calculation of risk. In each example, gestational dating is by ultrasound measure. Triple: AFP, uE3, hCG; Quad:
with the fetal image in sagittal plane and en- AFP, uE3, hCG, inhibin A; Combined: NT, PAPP-A, free beta-hCG; Serum Integrated: PAPP-A, AFP, uE3, hCG, inhibin A; Full Integrated:
NT, PAPP-A, AFP, uE3, hCG, inhibin A.
larged to take up about 75% of the view. The
image is digitally frozen and, by convention, Published and unpublished data from Prof. N. Wald, Wolfson Institute of Preventive Medicine (University of London, U.K.).
calipers are then placed on the inner edge of
the fetal skin and the outer edge of the spine than 2% of unaffected pregnancies have an Is There a Better Screening looking at what false positive rate is needed
at the back of the neck, perpendicular to the NT MoM ≥ 2.0, showing that an increased Option Available Now? to achieve a particular level of detection. For
plane of the spine. Technicians make the NT measurement is much less common In 1999, British epidemiologist Nicholas example, if we ask what false positive rate
NT measurement in tenths of millimeters than an elevated hCG level (Figure 2). Wald developed a prenatal screening test would result in an 85% detection rate when
and report it to the screening program re- for Down syndrome that integrated the best using the triple test, quad test, first trimester
sponsible for risk reporting, along with the First Trimester Screening: How Good Is It? markers available in the first and second screen, serum integrated test, and complete
fetal crown-rump length, which is used for First trimester screening performance using trimesters. The so-called “integrated test” integrated test, they would be 14%, 9%, 5%,
dating purposes. NT, PAPP-A, and free beta-hCG appears to is ideally done in concert with the two best 5%, and 1%, respectively. If the goal is a 90%
Typically, the NT measurement is in the be about an 85% detection rate for a 5% first trimester markers, NT and PAPP-A, detection rate, the respective false positive
range of 0.5 to 1.5 mm, and training and false positive rate, with detection rates re- and the second trimester quad markers. rates for the same tests would be 21%, 15%,
practice are required to achieve consistency ported in the literature ranging from 76% to Alternatively, the serum-only version of the 10%, 9%, and 2% (Figure 3).
and accuracy. In many ways, this measure- 92%. Neither NT alone (about 65% detec- integrated test is done without NT, using These relative results are quite reproduc-
ment must be treated as if it were a measure- tion rate for a 5% false positive rate) nor a PAPP-A plus the quad markers. This ap- ible, having been determined in both the
ment made in the laboratory, with similar serum marker combination alone (about proach is based on the simple concept that U.K. SURUSS trial and the U.S. FASTER
quality assurance and review. In addition, 63% detection rate for a 5% false positive if the markers are sufficiently independent trial. Also, at the First Brown Conference
NT increases naturally with gestational rate) in the first trimester is sufficiently indicators of risk, using all of the first and on the Integrated Test, held in March 2003,
age, at a rate of approximately 20% per good to be offered as an alternative to quad second trimester markers together will nec- a consortium of programs in Europe and
week, similar to the increases seen with AFP marker screening in the second trimester essarily produce a better screening test than Canada that have implemented the inte-
(15% per week), uE3 (25% per week), and and therefore should not be offered. using either the first or second trimester tests grated test reported that it was performing
PAPP-A (almost 50% per week). Therefore, The accumulated evidence indicates that alone. Free beta-hCG or hCG levels are not as expected.
normalization of the NT measurement the performance of first trimester screen- measured twice; rather, one or the other is With these data, it now seems that mark-
by comparison of the patient value to the ing with combined ultrasound and serum measured in the second trimester when its edly safer screening is possible. Rather than
median value for that day of gestation, as markers is in the same range as second performance is best. subjecting 5% or more of pregnant women
is done for all of the serum markers, is pos- trimester quad marker screening. Two re- The integrated test requires that a woman to the risks of CVS or amniocentesis, the rate
sible. The normalized value would then be cent population-based studies, SURUSS in have an NT measurement and a blood draw of diagnostic intervention can be reduced by
reported as a multiple of the population the U.K. and the FASTER Trial in the U.S., for PAPP-A measurement between 10 and as much as 75–80%, to only 1% or 2%. To
median, or MoM. were specifically designed to compare first 13 gestational weeks, and then return for a achieve such improvement in performance,
On average, NT is two times higher in and second trimester methods and neither second blood draw ideally at 15 or 16 weeks. the test must be done in two parts, with 2
Down syndrome as compared to unaffected showed a significant gain with one over the However, the second blood can be drawn to 6 weeks intervening before the result is
pregnancies; however, the distribution of NT other. Therefore, while first trimester screen- as late as 22 gestational weeks. The results reported. Amniocentesis, rather than CVS,
MoM values in unaffected pregnancies has a ing has the potential benefit of leading to of the NT and PAPP-A measurements are is offered to screen positive women so that
very narrow range of measurements. Conse- earlier diagnosis, it does not appear to be a not reported until they can be integrated diagnosis of Down syndrome, if present, is
quently, increased MoM values are relatively markedly better test than second trimester with the quad marker results from the sec- made at the same time in pregnancy as is
rare, making it a more powerful marker quad marker screening. Also, the question ond trimester blood sample into a single usual following second trimester screening.
than any of the serum markers discovered of widespread, low cost implementation of a estimate of risk. Alternatively, if the serum In addition, screening for open neural tube
so far. For example, the median for both first sonographic test, the requirement that early version of the integrated test is done, the test defects with AFP is part of the protocol.
trimester NT and second trimester hCG in diagnostic intervention by CVS be read- simply involves two separate blood draws, as
Down syndrome pregnancies is 2.0 MoM, ily available, and the continuing need for a described above. Guidelines to Consider
which is another way of saying that 50% of second trimester screen for open neural tube The performance of the integrated test In 2004, we are faced with a number of new
Down syndrome cases have an MoM ≥ 2.0. defects make the decision to choose screen- is markedly superior to that of either first choices in prenatal screening for Down
However, almost 10% of unaffected preg- ing a few weeks earlier than is now routinely trimester screening or second trimester se- syndrome and must decide how and when
nancies have an hCG MoM ≥ 2.0, while less available a less than optimal alternative. rum screening and can best be illustrated by to offer them. Rather than choosing from a

14 CLINICAL LABORATORY NEWS AUGUST 2004
ter Down’s syndrome screening. J Med Wapner R, Thom E, Simpson JL, et al. First-
Suggested Guidelines for Screen 2001;8:2–7.
Malone, FD, Wald NJ, Canick JA, et al. First-
trimester screening for trisomies 21 and
18. N Engl J Med 2003:349:1405–13. CLN
Down Syndrome Screening and second-trimester evaluation of risk
(FASTER) trial: principal results of the
Pregnant women should be informed that: NICHD multicenter Down syndrome Jacob Canick, PhD, is a
1. If NT measurement is available, the full integrated test offers the high- screening study. Am J Obstet Gynecol Professor in the Department
est screening performance for identification of Down syndrome preg- 2003;189:S56. of Pathology and Laboratory
nancy, and should be offered to all pregnant women. Malone FD, D’Alton ME, Berkowitz R, Medicine, Brown Medical
Canick JA. First trimester screening for School, and Director of the
2. If NT measurement is not available, the serum-only version of the inte- Division of Prenatal and Special Testing,
aneuploidy: research or standard of care?
grated test will provide the best screening performance and should be Department of Pathology, Women and
Am J Obstet Gynecol 2000;182;490–6.
offered. Infants Hospital, Providence, R.I. He is also
Palomaki GE, Knight GJ, McCarthy JE,
3. If a woman wants the earliest available prenatal diagnosis, and CVS is a consultant to Diagnostic Systems Labora-
Haddow JE, Donhowe JM. Maternal
available, the first trimester screen, combining NT and serum markers, tories, Webster, Tex., and with others, holds
serum screening for Down syndrome
should be offered. NT alone or serum markers alone should not be of- two patents on the use of unconjugated
in the United States: A 1995 survey. Am J
fered. estriol in prenatal screening. E-mail address:
Obstet Gynecol 1997;176:1046–51.
jcanick@wihri.org
4. If a pregnant woman does not present for prenatal care before 14 Wald NJ, Kennard A, Hackshaw A, McGuire
gestational weeks, the second trimester screening with quad mark- A. Antenatal screening for Down’s syn-
ers should be offered. Second trimester screening with only double or drome. J Med Screen 1997;4:181–246.
This article is available as an
triple markers should not be offered. Wald NJ, Watt HC, Hackshaw AK. Inte-
grated screening for Down’s syndrome 8 1/2” x 11” reprint on the
menu of tests offered as equal alternatives, ation the issues described above and provide on the basis of tests performed during
the first and second trimesters. N Engl J AACC Web site
we should try to offer the best test available, laboratorians with a possible preview of the
always keeping in mind that women have future of Down syndrome screening. Med 1999;341:461–7.
(www.aacc.org).
the right to accept or reject that test, or to Wald NJ, Rodeck C, Hackshaw AK, Walters
opt for an alternative. SUGGESTED READINGS J, Chitty L, Mackinson AM. First and Click on “Publications,”
But some issues still need to be addressed Canick JA, Lambert-Messerlian GM, Farina second trimester antenatal screening
before the medical community can come to A. General principles of second trimester for Down’s syndrome: the results of the “Clinical Laboratory News,”
agreement on which is the best test avail- maternal serum screening for Down serum, urine and ultrasound screening
then “Series Articles.”
able. Some investigators have stated that, syndrome. UpToDate, Clinical Reference study (SURUSS). J Med Screen 2003;10:
“Women won’t wait,” referring to the out- Library (on-line and on CD-ROM), 56–104.
look that first trimester screening is the best Wellesley, Mass, updated annually, cur-
test because it allows for the earliest possible rent version 12.1, 2004.
diagnosis. While there is no doubt that the Canick JA, Lambert-Messerlian GM, Fa-
earliest possible screening test would pro- rina A. Second trimester maternal serum
vide the best options for women and their screening for Down syndrome: clinical
babies, there are other factors to weigh. recommendations. UpToDate, Clinical
For example, it is reasonable to assume Reference Library (on-line and on CD-
that many women would prefer a test that ROM), Wellesley, Mass., updated annu-
exposes them to markedly less chance of los- ally, current version 12.1, 2004.
ing a wanted pregnancy by allowing them Canick JA, Lambert-Messerlian GM, Farina
to avoid the risks of CVS or amniocentesis. A. First trimester screening for Down
Given the trend toward delaying pregnancy syndrome. UpToDate, Clinical Reference
until women are in their 30s and 40s, it is Library (on-line and on CD-ROM),
critical that such options are known to preg- Wellesley, Mass., updated annually, cur-
nant women as they evaluate their choices. rent version 12.1, 2004.
Indeed, some professional societies in the Knight GJ, Palomaki GE, Neveux LM,
field are now considering new guidelines for Haddow JE, Lambert-Messerlian GM.
Down screening. The suggested guidelines Clinical validation of a new dimeric in-
offered here (see box) take into consider- hibin-A assay suitable for second trimes-

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CLINICAL LABORATORY NEWS AUGUST 2004 15