Joint SOGC-CCMG Clinical Practice Guideline
No. 265, September 2011
Use of a DNA Method, QF-PCR, in the Prenatal
Diagnosis of Fetal Aneuploidies
This Clinical Practice Guideline has been prepared by
the Genetics Committee of the Society of Obstetricians
and Gynaecologists of Canada (SOGC) and the Prenatal
Diagnosis Committee of the Canadian College of Medical
Geneticists (CCMG) and approved by the Executive and
Council of the SOGC and the Board of Directors of the
CCMG.
Valérie A. Désilets, MD, Montreal QC
PRINCIPAL AUTHORS
Vicky Siu, MD, London ON
Sylvie Langlois MD, Vancouver BC
David Skidmore, MD, Halifax NS
Alessandra Duncan, PhD, Montreal QC
Disclosure statements have been received from all members of
the committees.
SOGC GENETICS COMMITTEE
Alessandra Duncan, PhD, Montreal QC
Michael T. Geraghty, MD, Ottawa ON
Janet Marcadier, MSc, Ottawa ON
Tanya N. Nelson, PhD, Vancouver BC
R. Douglas Wilson, MD (Chair), Calgary AB
François Audibert, MD, Montreal QC
Jo-Ann Brock, MD, Halifax NS
June Carroll, MD, Toronto ON
Lola Cartier, MSc, CCGC, Montreal QC
Valérie A. Désilets, MD, Montreal QC
Alain Gagnon, MD, Vancouver BC
Jo-Ann Johnson, MD, Calgary AB
Sylvie Langlois, MD, Vancouver BC
Lynn Murphy-Kaulbeck, MD, Moncton NB
Nanette Okun, MD, Toronto ON
Melanie Pastuck, RN, Cochrane AB
CCMG PRENATAL DIAGNOSIS COMMITTEE
Sylvie Langlois, MD (Chair), Vancouver BC
David Chitayat, MD, Toronto ON
Isabelle DeBie, MD, Montreal QC
Suzanne Demczuk, PhD, Saskatoon SK
Key Words: Quantitative fluorescent polymerase chain reaction
(QF-PCR), fetal chromosomal abnormalities, prenatal diagnosis
Abstract
Objective: To provide Canadian health care providers with current
information on the use of quantitative fluorescent polymerase
chain reaction (QF-PCR) or equivalent technology in the prenatal
diagnosis of fetal chromosomal abnormalities.
Options: Over the last few decades, prenatal diagnosis of fetal
chromosomal abnormalities has relied on conventional cytogenetic
analysis of cultured amniocytes, chorionic villi, or fetal blood.
In the last few years, the clinical validity of a newer technique,
QF-PCR, to detect the common aneuploidies has been reported
by a number of investigators. This technique has the advantage of
providing rapid results for the diagnosis or exclusion of aneuploidy
in chromosomes 13, 18, 21, X or Y. It is now possible to choose
standard chromosome analysis or QF-PCR for the prenatal
diagnosis of chromosomal abnormalities, or to perform both tests,
depending on the clinical indication for testing. This document
reviews the clinical utility of QF-PCR and makes recommendations
for its use in the care of Canadian patients.
Evidence: Medline and PubMed were searched for articles
published in English between January 2000 and December 2010
that presented data on the use of QF-PCR versus standard
cytogenetic analysis of prenatal samples. A second search was
done to identify publications in English that provided results
of cytogenetic analysis performed on prenatal samples for
women at an increased risk of fetal aneuploidy because of
maternal age, abnormal prenatal screening results, or fetal soft
ultrasound markers suggestive of an increased risk of aneuploidy.
This document reflects emerging clinical and scientific advances on the date issued, and is subject to change. The information
should not be construed as dictating an exclusive course of treatment or procedure to be followed. Local institutions can dictate
amendments to these opinions. They should be well documented if modified at the local level. None of these contents may be
reproduced in any form without prior written permission of the SOGC.
SEPTEMBER JOGC SEPTEMBRE 2011 l 955
Joint SOGC-CCMG Clinical Practice Guideline
Publications were included if they provided detailed information
on the abnormalities detected, regardless of whether or not rapid
aneuploidy screening was undertaken.
Results were restricted to systematic reviews, randomized
controlled trials, and relevant observational studies. Grey
(unpublished) literature was identified through searching the
websites of health technology assessment and health technology
assessment-related agencies, clinical practice guideline
collections, clinical trial registries, and national and international
medical specialty societies.
Values: The quality of evidence was rated using the criteria described
in the Report of the Canadian Task Force on Preventive Health
Care (Table 1).
Benefits, harms, and costs: This guideline promotes the use of a
rapid aneuploidy DNA test for women at increased risk of having
a pregnancy affected by a common aneuploidy. This will have
the benefit of providing rapid and accurate results to women at
increased risk of fetal Down syndrome, trisomy 13, trisomy 18,
sex chromosome aneuploidy or triploidy. It will also promote
better use of laboratory resources and reduce the cost of prenatal
diagnosis. However, a small percentage of pregnancies with a
potentially clinically significant chromosomal abnormality will
remain undetected by QF-PCR but detectable by conventional
cytogenetics.
Recommendations
1. QF-PCR is a reliable method to detect trisomies and should
replace conventional cytogenetic analysis whenever prenatal
testing is performed solely because of an increased risk of
aneuploidy in chromosomes 13, 18, 21, X or Y. As with all tests,
pretest counselling should include a discussion of the benefits
and limitations of the test. In the initial period of use, education for
health care providers will be required. (II-2A)
2. Both conventional cytogenetics and QF-PCR should be
performed in all cases of prenatal diagnosis referred for a
fetal ultrasound abnormality (including an increased nuchal
translucency measurement > 3.5 mm) or a familial chromosomal
rearrangement. (II-2A)
3. Cytogenetic follow-up of QF-PCR findings of trisomy 13 and 21 is
recommended to rule out inherited Robertsonian translocations.
However, the decision to set up a back-up culture for all cases
that would allow for traditional cytogenetic testing if indicated by
additional clinical or laboratory information should be made by
each centre offering the testing according to the local clinical and
laboratory experience and resources. (III-A)
4. Other technologies for the rapid detection of aneuploidy may
replace QF-PCR if they offer a similar or improved performance
for the detection of trisomy 13, 18, 21, and sex chromosome
aneuploidy. (III-A)
J Obstet Gynaecol Can 2011;33(9):955–960
ABBREVIATIONS
FISH
fluorescence in situ hybridization
QF-PCR quantitative fluorescent polymerase chain reaction
956 l SEPTEMBER JOGC SEPTEMBRE 2011
INTRODUCTION
O
ver the last few decades, prenatal diagnosis of
fetal chromosomal abnormalities has relied on
conventional cytogenetic analysis of cultured amniocytes,
chorionic villi, or fetal blood. FISH on uncultured
amniocytes or chorionic villi has been added in some cases
for the rapid detection of aneuploidy in chromosomes
13, 18, 21, X and Y. In the last few years, the clinical
utility of using QF-PCR to detect common aneuploidies
has been reported by a number of investigators. These
new molecular techniques for the prenatal detection of
chromosomal aneuploidy are reviewed in more detail in a
Society of Obstetricians and Gynaecologists of Canada
technical update.1 QF-PCR is a PCR-based technique
that consists of amplifying polymorphic markers located
on the chromosomes of interest to determine the
number of copies of those chromosomes present per
cell. The advantages of QF-PCR are that it requires a
small sample and allows automation of the procedure,
providing a rapid turnaround time at a lower cost than
conventional cytogenetics. Moreover, diagnostic testing
with QF-PCR eliminates the unexpected or incidental
identification of rare chromosomal abnormalities of
uncertain significance, whereas G-banding karyotyping
can yield results with a low predictive value for abnormal
phenotype because of the detection of mosaicism
and de novo balanced rearrangements.2 Some authors
have suggested that because of its high sensitivity and
specificity for the detection of the common aneuploidies,
QF-PCR should replace conventional chromosome
analysis as the method of prenatal diagnostic testing
in pregnancies at an increased risk for fetal trisomy 18
or trisomy 21 because of maternal age or an abnormal
prenatal screening test result; conventional chromosome
analysis would continue to be used for cases determined
to be at increased risk for fetal chromosomal abnormality
because of a fetal structural anomaly detected by
ultrasound.3–5
PERFORMANCE OF QF-PCR IN DETECTING
NON-MOSAIC TRISOMY 13, 18, 21, TRIPLOIDY,
AND SEX CHROMOSOMAL ANEUPLOIDY
To assess the performance of QF-PCR in detecting nonmosaic trisomy 13, 18, 21, triploidy, and sex chromosomal
aneuploidies, data were collected from the relevant
publications to answer three main questions:
1. What percentage of cases cannot be reported because
either the PCR failed or the results are inconclusive
because of maternal cell contamination?
Use of a DNA Method, QF-PCR, in the Prenatal Diagnosis of Fetal Aneuploidies
Table 1. Key to evidence statements and grading of recommendations, using the ranking of the Canadian Task Force
on Preventive Health Care
Quality of evidence assessment*
Classification of recommendations†
I:
A. There is good evidence to recommend the clinical preventive action
Evidence obtained from at least one properly randomized
controlled trial
II-1: Evidence from well-designed controlled trials without
randomization
B. There is fair evidence to recommend the clinical preventive action
II-2: Evidence from well–designed cohort (prospective or
retrospective) or case–control studies, preferably from
more than one centre or research group
C. The existing evidence is conflicting and does not allow to make a
recommendation for or against use of the clinical preventive action;
however, other factors may influence decision-making
II-3: Evidence obtained from comparisons between times or
places with or without the intervention. Dramatic results in
uncontrolled experiments (such as the results of treatment with
penicillin in the 1940s) could also be included in this category
D. There is fair evidence to recommend against the clinical preventive action
III:
L. There is insufficient evidence (in quantity or quality) to make
a recommendation; however, other factors may influence
decision-making
Opinions of respected authorities, based on clinical experience,
descriptive studies, or reports of expert committees
E. There is good evidence to recommend against the clinical preventive
action
*The quality of evidence reported in these guidelines has been adapted from The Evaluation of Evidence criteria described in the Canadian Task Force on
Preventive Health Care.23
†Recommendations included in these guidelines have been adapted from the Classification of Recommendations criteria described in the Canadian Task Force
on Preventive Health Care.23
2. Of the cases reported, are there any false-positive
diagnoses defined as a pregnancy with a normal
karyotype but in which the QF-PCR result was
reported to be abnormal?
3. Of the cases reported, are there any false-negative
diagnoses by QF-PCR of non-mosaic trisomy 13,
18, 21, triploidy, and sex chromosomal aneuploidies?
The data extracted from relevant publications are presented
in Table 2.
In total, 79 556 prenatal samples were analyzed. A small
proportion (1.3%) of samples could not be reported,
predominantly because of maternal cell contamination
that caused an inconclusive result. In contrast, cytogenetic
analysis was unsuccessful because of culture failure
of amniocytes in 0.12% to 0.3% of cases.9,10,15,16 The
performance of the QF-PCR test is dependent on the
number of markers analyzed per chromosome. In the
original studies, a number of cases remained uninformative
for one or more chromosomes. This problem seems to
have been resolved by the typing of additional markers,
as shown in a large 2009 study.15 Enough polymorphic
markers must be typed for each chromosome of interest
to ensure accurate determination of copy number.
Among all studies reviewed, no false-positive diagnoses
were made. All cases found to be abnormal by QF-PCR
were abnormal on conventional cytogenetic analysis,
although in 5 cases of sex chromosomal aneuploidy, an
incorrect diagnosis was made. Four of these cases were
interpreted as 47,XXX by QF-PCR but were diagnosed by
cytogenetic analysis as mosaic 45,X / 46,XX,15 and one case
was interpreted as 47,XXY by QF-PCR but was mosaic 47,
XXY/ 46,XX by cytogenetic analysis.7 Among the 79 556
prenatal samples compiled in this review, there were 2674
cases of non-mosaic trisomy 13, 18, or 21 or triploidy. Two
false-negative cases of trisomy were reported in one study
that analyzed only 2 markers per chromosome.12 There
were actually no other false-negatives, because the 7 cases
not diagnosed by QF-PCR were not reported as normal but
were uninformative because of maternal cell contamination.
There were 328 cases of non-mosaic sex chromosomal
aneuploidy. Fifteen cases were missed. The accuracy of
the diagnosis of sex chromosomal aneuploidy appears to
depend on the panel of X chromosome markers used in
the analysis by each investigator. Cirigliano et al. missed one
case early in their series and none after additional markers
were added to the panel.15 This suggests that with the
currently available panels non-mosaic sex chromosomal
aneuploidy cases can be reliably detected.
In summary, 98.7% of samples analyzed by QF-PCR have
conclusive results, with no false-positive or false-negative
diagnosis of non-mosaic trisomy for chromosomes 13, 18,
or 21 or triploidy. Although no false-positive diagnosis was
made for sex chromosomal aneuploidy, some earlier studies
reported false-negative results. Currently, these would
seem less likely to occur because of the implementation of
additional polymorphic markers.
SEPTEMBER JOGC SEPTEMBRE 2011 l 957
Joint SOGC-CCMG Clinical Practice Guideline
Table 2. Performance of QF-PCR
Number
and type of
specimens
Sample failure/
not tested or
UI, %
Number of cases
detected / number
of actual nonmosaic autosomal
trisomies and
triploidies
Levett et al. 20016
5097 AF
0.1 / 2
89/89
0
16/20
0
Schmidt et al. 20017
662 AF
0/0
14/15
1 case of
T18 was UI
0
5/5*
0
Bili et al. 20028
1020 AF
0 / 1.2
16/19
3 cases were UI
0
0/0
0
7720
(6147AF,
1552 CVS
21 FBS)
0.09 / 2.1
437/437
0
N/A
N/A
Caine et al. 200510
10253AF
2.9
429/429
0
NA
NA
Brown et al. 2006
687 AF
0 / 2.5
14/14
0
1/3
Only 2 markers
on X chr
0
Kozlowski et al. 200612
4692 AF
0.06 / 0.15
71/73
2 cases missed at
the beginning when
only two markers
per chr used.
0
2/8
0
Kagan et al. 20074
3854 AF
None reported
202/202
0
14/14
0
Onay et al. 200813
576 AF
0 / 2.9
15/15
0
4/4
0
2906
(142 CVS,
2764 AF)
0 / 0.26 for AF
110/110
0
20 / 20
0
37544 AF
4687 CVS
0.05 / 0.82
1287/1290†
3 cases were UI
0
265/267†‡
1 abnormality
missed;
1 UI MCC
0
Reference
Mann et al. 20049
11
Putzova et al. 2008
14
Cirigliano et al. 200915
False
positives for
trisomy or
triploidy
Number of
cases detected /
number of actual
non-mosaic sex
chromosomal
aneuploidies
False
positives
for sex
chromosomal
aneuploidy
AF: amniotic fluid; chr: chromosome; CVS: chorionic villus sampling; FBS: fetal blood sample; MCC: maternal cell contamination;
UI: reported as uninformative because of MCC or uninformative markers.
*1 case with a cytogenetic diagnosis of 47,XXY/46,XX was diagnosed by QF-PCR as 47,XXY.
†4 cases of 48,XXY+21, 3 cases of 48,XXY+18 and 1 case of 68,XX are included in the total numbers of both autosomal aneuploidies/triploidies and sex
chromosomal aneuploidies.
‡4 cases with a cytogenetic diagnosis of 45,X/46,XX were diagnosed by QF-PCR as 47,XXX.
Residual Risk of a Chromosomal
Abnormality Given a Negative QF-PCR Result
Although QF-PCR has been shown to be a reliable method
to detect non-mosaic cases of trisomy 13, 18, and 21 and sex
chromosomal aneuploidy, it is less reliable in the detection
of mosaic cases. Cirigliano et al. reported diagnosis of 45
out of the 72 mosaic cases in their series.15 Furthermore,
QF-PCR is not designed to detect trisomies other than 13,
18, and 21 and will not detect most unbalanced chromosomal
abnormalities, nor will it detect balanced rearrangements.
Therefore, a patient with a negative QF-PCR result has a
residual risk of having a pregnancy with a chromosomal
958 l SEPTEMBER JOGC SEPTEMBRE 2011
abnormality detectable on conventional chromosome
analysis. To calculate this risk in pregnancies having fetal
chromosome analysis for abnormal prenatal screening,
maternal age or a soft marker of aneuploidy on ultrasound,
prenatal series that reported all prenatal cytogenetic results
were included in this analysis, regardless of whether QF-PCR
was performed. When it was not performed, it was assumed
that all cases of non-mosaic trisomy 13, 18, 21, triploidy,
and sex chromosomal aneuploidy would be detected by
QF-PCR. The residual risk was defined as the number of
cases having a cytogenetic abnormality not detected by
QF-PCR over the number of cases with normal results
Use of a DNA Method, QF-PCR, in the Prenatal Diagnosis of Fetal Aneuploidies
Table 3. Residual risk of a chromosomal abnormality given normal QF-PCR results
Reference
Caron et al. 199917
Ryall et al. 2001 Abn
screen
18
Caine et al. 200510
Kozlowski et al. 2006
Kagan et al. 2007
Number
of cases
expected to
have negative
QF-PCR
results
Number of
abnormal of
low, unknown
or high risk not
detectable
by QF-PCR
Residual
risk for low,
unknown,
high, %
Number of
previously unknown
inherited balanced
rearrangements
or markers
Total
residual
risk, %
Number of
Samples
Number of
cases with
abnormal
detectable
by QF-PCR
024 901
0309
24 592
087
0.35
050
0.550
02737
0104
2633
006
0.23
019
0.950
111 510
3739
107 771
499
0.46
327
0.770
4404
0035
4369
021
0.48
016
0.856
12
3854
0216
3638
023
0.63
011
0.930
Speevak et al. 20082
3706
0088
3618
015
0.41
009
0.660
Sparkes, et al. 2008
5237
0103
5134
020
0.38
011
0.590
156 349
4594
151 755
671
0.44
443
0.730
Total
4
19
on QF-PCR.12 This residual risk was calculated for
chromosomal abnormalities associated with a low,
unknown, or high risk of abnormal outcome. Chromosomal
abnormalities associated with a low risk included de
novo apparently balanced rearrangements and cases in
which parents were unavailable for testing. Chromosomal
abnormalities of unknown risk included mosaic cases and de
novo marker chromosomes. Chromosomal abnormalities
associated with a high risk included other trisomies and
unbalanced structural rearrangements. Inherited balanced
chromosomal rearrangements or markers were considered
to confer no increased risk and were included in the total
residual risk. Table 3 provides the compiled data. Given a
negative QF-PCR result, the residual risk for a chromosomal
abnormality of low, high, or unknown risk is 0.44% or 1 in
227. If inherited balanced chromosomal rearrangements or
markers are included, the risk is 0.73% (1 in 137).
OTHER TECHNOLOGIES
FISH has been shown to be an accurate method to detect
fetal aneuploidy, and commercial kits are available to test
for trisomy 13, 18, 21, and sex chromosome aneuploidies.
However, QF-PCR has the advantage over FISH of being
much less expensive and of allowing the simultaneous
processing of a much larger number of samples.20
Although not as extensively studied as QF-PCR, multiplex
ligation-dependent probe amplification has been shown
to be a reliable rapid aneuploidy detection method, with
100% sensitivity and 100% specificity for the diagnosis
of aneuploidies of chromosomes X, Y, 13, 18, and 21
in a prospective cohort study of 4585 women.21 This
technology does not detect cases of triploidy. However,
such cases would be expected to have associated ultrasound
findings, and conventional cytogenetic analysis would be
indicated.
The field of molecular genetics is rapidly evolving.
Although currently QF-PCR would be considered the
technology of choice for rapid aneuploidy detection, it
is likely that other platforms will be developed that may
have the capacity to detect additional abnormalities such
as cases of segmental aneuploidies. For example, a bead
array approach with bacterial artificial chromosomes—
containing microbeads with probes for the aneuploidies
and microdeletion syndromes has been developed and
validated on a small number of samples. Results indicate
that this approach is a rapid and reliable test for common
aneuploidies and microdeletions.22
Recommendations
1. QF-PCR is a reliable method to detect trisomies
and should replace conventional cytogenetic
analysis whenever prenatal testing is performed
solely because of an increased risk of aneuploidy in
chromosomes 13, 18, 21, X or Y. As with all tests,
pretest counselling should include a discussion of
the benefits and limitations of the test. In the initial
period of use, education for health care providers
will be required. (II-2A)
2. Both conventional cytogenetics and QF-PCR should
be performed in all cases of prenatal diagnosis
referred for a fetal ultrasound abnormality
(including an increased nuchal translucency
measurement > 3.5 mm) or a familial chromosomal
rearrangement. (II-2A)
SEPTEMBER JOGC SEPTEMBRE 2011 l 959
Joint SOGC-CCMG Clinical Practice Guideline
3. Cytogenetic follow-up of QF-PCR findings of
trisomy 13 and 21 is recommended to rule out
inherited Robertsonian translocations. However,
the decision to set up a back-up culture for all cases
that would allow for traditional cytogenetic testing
if indicated by additional clinical or laboratory
information should be made by each centre offering
the testing according to the local clinical and
laboratory experience and resources. (III-A)
4. Other technologies for the rapid detection of
aneuploidy may replace QF-PCR if they offer a
similar or improved performance for the detection
of trisomy 13, 18, 21, and sex chromosome
aneuploidy. (III-A)
Glossary
Aneuploidy: A chromosome number that is not an exact multiple of
23, usually resulting from a meiotic non-disjunction error in the
production of gametes.
Chromosome: A linear structure containing a single strand of DNA.
A human normally has 46 chromosomes, in 23 pairs.
DNA: The molecule that encodes genes.
FISH: Fluorescence in situ hybridization.
Karyotype: The chromosome constitution of an individual, or the
photomicrograph of an individual’s chromosomes, systematically
arranged in 23 pairs.
Monosomy: The absence of a single chromosome.
Mosaicism: The presence of 2 or more genetically different cell lines
in an individual or tissue.
Numerical chromosome aberration: A chromosome number which
is not 46.
Structural chromosome aberration: A chromosome number of 46 in
which segment(s) of chromosome(s) are missing (deleted),
extra (inserted) or rearranged (translocated or inverted).
Triploidy: A chromosome number of 69 (3 copies of each
chromosome).
Trisomy: The presence of an extra chromosome.
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