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Obstetrics & Gynecology 2003;101:856-861
© 2003 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Can Amnio–Polymerase Chain Reaction Alone Replace Conventional Cytogenetic Study for Women With Positive Biochemical Screening for Fetal Down Syndrome?

Wing Cheong Leung, MRCOG, Elizabeth T. Lau, PhD, Terence T. Lao, MD, FRCOG and Mary Hoi Yin Tang, FRCOG

From the Department of Obstetrics and Gynaecology, Queen Mary Hospital, University of Hong Kong; and Prenatal Diagnostic and Counselling Department, Tsan Yuk Hospital, Hong Kong, People’s Republic of China.

Address reprint requests to: Wing Cheong Leung, MRCOG, Senior Medical Officer, Department of Obstetrics & Gynaecology, Queen Mary Hospital, 102, Pokfulam Road, Hong Kong, People’s Republic of China; E-mail: leungwc65{at}hotmail.com.


   ABSTRACT
TOP
ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE: To determine whether amnio–polymerase chain reaction (amnio-PCR) can replace conventional cytogenetic study for confirming the karyotype of fetuses in women with positive biochemical screening for fetal Down syndrome.

METHOD: To check the accuracy of this technique in our laboratory, we first compared the amnio-PCR results with those of conventional cytogenetic study in 235 patients referred from June 1999 to December 2001 for prenatal diagnosis in a referral center in Hong Kong. We then reviewed the results of 1526 amniotic fluid cultures performed for positive fetal Down syndrome screening between January 1997 and December 2001 and classified them as detectable or not detectable by amnio-PCR, using the assumption that we had replaced conventional cytogenetic study with amnio-PCR.

RESULTS: The 235 amnio-PCR results were all informative, without a false-positive or false-negative result. Of the 1526 cases with positive fetal Down syndrome screening and no ultrasound abnormalities, only two cases of sex chromosome abnormalities and two cases of marker chromosomes would have been missed if conventional cytogenetic study had been replaced by amnio-PCR.

CONCLUSION: Amnio-PCR can be an alternative to conventional cytogenetic study for women with positive biochemical screening for fetal Down syndrome and no demonstrable fetal structural abnormality.

Second-trimester maternal serum screening for fetal Down syndrome has become an established practice in many countries.1 The use and acceptability of screening with human chorionic gonadotrophin, alpha-fetoprotein, and/or unconjugated estriol has been demonstrated by numerous large prospective intervention studies.1 Serum screening generates a ratio that characterizes the risk of Down syndrome in the fetus. Women being assigned a risk above an arbitrary cutoff are designated screen positive and offered amniocentesis for a karyotype. Conventional cytogenetic study of the cultured amniocytes is performed, and a full karyotype report is available within 2 to 3 weeks. In addition to chromosome 21, all the other chromosomes are also assessed.

Von Eggeling et al2 devised a polymerase chain reaction (PCR)–based test that allowed rapid detection of trisomy 21 in less than 24 hours. This technique can be applied to deoxyribonucleic acid (DNA) from uncultured amniotic fluid cells that have been amplified with small tandem repeat markers using the PCR technique and fluorescence-labeled primers (amnio-PCR).3 The small tandem repeat markers specific for chromosome 21 will only be able to detect aneuploidy of chromosome 21. In a masked prospective study where 2167 pregnant women were offered amniocentesis, Verma et al4 showed that the rapid test was informative in 99.6% of cases and there were no false-positive or false-negative diagnoses of Down syndrome.

We have performed amnio-PCR for various indications since 1999 in our Prenatal Diagnostic and Counselling Department. In all cases, the diagnosis was confirmed by conventional cytogenetic study. Positive Down syndrome screening is the second most common indication for amnio-PCR, the first being ultrasound abnormalities.

Our research question is whether amnio-PCR can replace conventional cytogenetic study for women with only positive biochemical screening for fetal Down syndrome. In other words, we want to assess the likely outcome if we were to change to a policy of PCR analysis without conventional cytogenetic study for all of the amniocentesis samples from women with positive biochemical screening for fetal Down syndrome and no fetal abnormality detected on ultrasound examination. The potential advantage of this approach is the relief of women’s anxiety during the 2- to 3-week waiting time for the conventional cytogenetic study report.5 The potential disadvantage of this approach is that other chromosomal abnormalities apart from aneuploidy in chromosome 21 would not be detected. It would be important to know how many of these undetectable chromosomal abnormalities are clinically significant if the corresponding ultrasound examination does not show any fetal abnormality.


   MATERIALS AND METHODS
TOP
ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The data in this study were obtained from the database of the Prenatal Diagnostic and Counselling Department, Tsan Yuk Hospital, which is one of the referral centers for Hong Kong. We first studied all cases of amnio-PCR performed as a rapid test for prenatal diagnosis from June 1999 to December 2001. During this period, PCR was performed as an adjunct to conventional cytogenetic study at the discretion of the individual obstetrician. There was general agreement that amnio-PCR would be offered only if the results were likely to alter the management of the individual case. For the purpose of the present study, the indications for amniocentesis were categorized in a hierarchical fashion as follows: 1) ultrasound abnormalities, 2) positive Down syndrome screening, 3) advanced maternal age, 4) previous child with chromosomal abnormalities, and 5) others. If one or more ultrasound abnormalities were reported, the patient was classified as having ultrasound abnormalities irrespective of the Down syndrome screening result or maternal age. Ultrasound abnormalities refer to one or more fetal structural abnormalities, soft ultrasound markers, or evidence of fetal growth restriction (estimated fetal weight less than the tenth centile in local growth chart). Positive Down syndrome screening refers to an assigned risk of one in 250 or greater by assay of alpha-fetoprotein and human chorionic gonadotrophin between 15 and 20 weeks’ gestation. Advanced maternal age is defined as 35 years or older at the expected date of delivery.

Amnio-PCR was performed on uncultured amniocytes using small tandem repeat markers for chromosomes 13, 18, 21, X, and Y, with the choice of the chromosome to be tested dependent on the indications for amniocentesis. Cells from 2 to 3 mL of amniotic fluid were used for DNA preparation by a modified alkaline lysis method. Amniocytes were lysed in 20 µL of 0.2-mol/L potassium hydroxide, added with proteinase K (Roche Molecular Biochemicals, Mannheim, Germany) to a final concentration of 200 µg/mL, and incubated at 65C for 10 minutes. The solution was then neutralized with an equal volume of buffer containing 0.5-mol/L Tris (pH 8.5) and 0.2-mol/L hydrogen chloride and heated at 95C for 10 minutes. Deoxyribonucleic acid (2 µL) in the supernatant was then used for a single-tube multiplexed PCR of 10-µL volume using primers from small tandem repeat markers D21S1411, D21S1412, and D21S14146 labeled with IRD 700 or IRD 800 dye (LI-COR Inc., Lincoln, NE). Electrophoresis was performed on a LI-COR 4200 automatic DNA sequencer, and amplified products were analyzed with GeneImager software (LI-COR). All cases were followed by conventional cytogenetic studies. The cytogenetic results were classified as those detected or not detected by amnio-PCR.

We reviewed retrospectively the results of all amniotic fluid cultures performed between January 1997 and December 2001 with the indication of positive Down syndrome screening from our database. The cytogenetic results were classified as being detectable or undetectable by amnio-PCR, with the assumption that we were using a policy of PCR analysis not followed by conventional cytogenetic study for all of the amniocentesis samples from women with positive biochemical Down syndrome screening and no demonstrable fetal abnormality.


   RESULTS
TOP
ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We had performed 235 amnio-PCR tests from June 1999 to December 2001. Amnio-PCR was informative in all cases. Table 1Go shows the various indications in a hierarchical fashion, the number of abnormal karyotypes detected, and whether they were detected or not by amnio-PCR. The presence of ultrasound abnormalities was the most common indication (56.2%), followed by positive Down syndrome screening (28.9%), advanced maternal age (6.8%), previous child with chromosomal abnormalities (all were trisomies) (2.6%), and others (5.5%). There were altogether 63 abnormal karyotypes. Amnio-PCR could detect 57 of them (90.5%) (Table 2Go). There were no false-positive or false-negative amnio-PCR results regarding the diagnosis of aneuploidy in chromosomes 13, 18, 21, X, and Y. Amniotic fluid culture failure occurred in two cases. The two amniocenteses were performed at 33 and 38 weeks, respectively, both because of ultrasound abnormalities. Amnio-PCR detected trisomy 21 in one case and no evidence of trisomy 13/18/21 in the other.


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  		Table 1. Indications for the 235 Amniocenteses (With Polymerase Chain Reaction Performed), Number of Abnormal Karyotypes Detected and Whether They Were Detected by Polymerase Chain Reaction
 

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  		Table 2. Abnormal Karyotypes Detected by Polymerase Chain Reaction
 
There were six cases of abnormal karyotypes not detected by amnio-PCR. None of them would be detectable by the small tandem repeat markers for chromosome 13, 18, 21, X, or Y used in this study. Four of them had ultrasound abnormalities, and the clinical outcome is shown in Table 3Go. Of the two cases of no ultrasound abnormalities (Table 4Go), one was balanced Robertsonian translocation inherited from the father, with no clinical significance. Amniocentesis was performed because the father was a known balanced Robertsonian translocation carrier. The other case (47,XX,+mar) was clinically significant because the de novo marker was found to be 15q, involving the Prader–Willi/Angelman syndrome chromosome region. The clinical outcome was a phenotypically normal live birth but with the potential of having mental and developmental delay. Amniocentesis was performed because of positive Down syndrome screening. Thus this was the only case that would have been missed if we had adopted a policy of PCR analysis not followed by conventional cytogenetic study for the amniotic fluid samples obtained for the indication of positive biochemical Down syndrome screening.


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  		Table 3. Abnormal Karyotypes Not Detected by Polymerase Chain Reaction, Associated With Ultrasound Abnormalities (n = 4)
 

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  		Table 4. Abnormal Karyotypes Not Detected by Polymerase Chain Reaction, and Not Associated With Ultrasound Abnormalities (n = 2)
 
The results of the retrospective review of all of the 1526 cases with amniotic fluid cultures performed between January 1997 and December 2001 with the indication of positive Down syndrome screening are shown in Table 5Go. There were 53 cases of trisomy 21 (3.5%), four cases of trisomy 18 (0.3%), one case of trisomy 13 (0.1%), three cases of sex chromosome abnormalities (0.2%), and four cases of other chromosomal abnormalities (0.3%).


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  		Table 5. Amniotic Fluid Standard Karyotype Analysis for Positive Down Syndrome Screening From 1997 to 2001
 
If we had adopted the policy of PCR analysis not followed by conventional cytogenetic study for all of the amniocentesis samples from women with positive biochemical Down syndrome screening and no demonstrable fetal abnormalities, six cases of chromosomal abnormalities (six of 65 [9.2%]) would have been missed. The nature of the chromosomal abnormalities and the clinical outcome of these six cases are shown in Table 6Go. We had not included a case of mosaic trisomy 21, 47,XX,+21/46,XX(10:24), because technically this could be detected by amnio-PCR or reported as inconclusive, depending on the level of mosaicism. The six cases that would have been completely missed by amnio-PCR included two of marker chromosomes, two of balanced Robertsonian translocation, and two of sex chromosome abnormalities. One case of marker chromosome (15q) was the one described previously (Table 4Go). The other one was a de novo marker of unknown nature with a normal phenotype. Regarding the two cases of balanced Robertsonian translocation, one was paternal in origin and one was de novo; both were of no clinical significance. The two cases of sex chromosome abnormalities had completely different outcomes. For the 47,XXX/45,X(31:10) case, the parents accepted the diagnosis and continued with the pregnancy. A baby girl was born at 41 weeks’ gestation with normal phenotype. For the 47,XXY case, the parents did not accept their child having Klinefelter syndrome, and termination of pregnancy was performed. There is another case of sex chromosome abnormality (45,X), but it would not be missed under the proposed policy because it is associated with hydropic changes on ultrasound examination.


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  		Table 6. Abnormal Karyotypes From Amniocentesis for Positive Down Syndrome Screening That Would Be Missed Under the New Approach
 

   DISCUSSION
TOP
ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Amnio-PCR has been shown to diagnose chromosome aneuploidy accurately.3,4,7,8 Its major advantage is fast reporting, within 24–48 hours. Relative to the other fast reporting technique of amniotic fluid interphase fluorescence in situ hybridization, amnio-PCR is less labor intensive. Many centers are already terminating pregnancy based on abnormal amnio-PCR results without waiting for the conventional cytogenetic confirmation.9 The potential use of amnio-PCR as a stand-alone test for indications such as positive serum Down syndrome screening and advanced maternal age has been suggested.8 For cases of no ultrasound abnormalities, one might worry that chromosomal abnormalities apart from trisomy 21 would be missed by this approach. However, we must realize that Down syndrome screening is a program designed to primarily detect Down syndrome. Therefore, in principle and in practice, follow-up testing with amnio-PCR alone in patients with positive Down syndrome screening would have realistically fulfilled the expectations of the patients and obstetricians.

Active research is being done on how to achieve these realistic expectations of patients, providers, and policy makers for prenatal testing.10 In fact, all of the screening methods (age, history, biochemical) are backed up by a high-level ultrasound examination to look for fetal abnormalities. Therefore, amnio-PCR as a stand-alone test is not literally alone. In our database of 1526 cases with positive Down syndrome screening, all four cases of trisomy 18 and one case of trisomy 13 were associated with major ultrasound abnormalities. Even under the proposed policy, these cases would have been identified because conventional cytogenetic study would be performed in the presence of ultrasound abnormalities. Furthermore, they would also be diagnosed by amnio-PCR using small tandem markers for chromosomes 13 and 18.

The cases that are likely to have been missed by this approach are the sex chromosome abnormalities, balanced translocations, and marker chromosomes that are not associated with ultrasound abnormalities. Balanced translocations with normal phenotypes are basically clinically insignificant, and there is no consensus for the management of the other conditions. Counseling regarding sex chromosome abnormalities is often difficult. The presence of marker chromosomes is an even more difficult area for counseling, particularly in the absence of phenotypic abnormalities.11 The revelation to the parents of these chromosomal abnormalities found incidentally in a program designed to primarily detect Down syndrome could lead to unnecessary anxiety and even unwarranted termination of pregnancy, as parental reactions can vary greatly. The outcome can be unpredictable, with some parents requesting termination of pregnancy and others accepting the diagnosis, as illustrated by the two cases in our study.

The adoption of a policy of PCR analysis alone for the amniocentesis samples from women with only positive biochemical Down syndrome screening has two advantages. The first and obvious one is the fast reporting. There is a general belief that the fast reporting of normal amnio-PCR results can relieve parental anxiety while they are awaiting the final report from the cytogenetic study. However, a recent randomized controlled trial showed that amnio-PCR did not alleviate anxiety in women who are screen positive for Down syndrome screening.5 One possible explanation is that the woman, although told that the amnio-PCR result is normal, is still having a significant degree of anxiety while waiting for the confirmation by the full karyotype report. This anxiety might be alleviated if the amnio-PCR report is considered to be final. Such an approach will also eliminate the problems encountered with culture failure and the dilemma of repeat amniocentesis, fetal blood sampling, or assuming the result to be normal. A prospective study on the anxiety levels of these women, with one group randomized to having amnio-PCR as a stand-alone test and the other group to having amnio-PCR followed by conventional cytogenetic study, is being planned in our hospital. The second advantage is cost savings. Instead of adding the cost of amnio-PCR on top of that of conventional cytogenetic study, the cost of the latter can be saved by the amnio-PCR–alone approach. In this age of ever-escalating costs in the provision of health care, especially in a government-funded public medical care system as in Hong Kong, the savings can be redirected to enhance existing programs or fund new ones, thus maximizing the effect of limited resources.

In conclusion, we think that amnio-PCR can replace conventional cytogenetic study for women with positive biochemical screening for fetal Down syndrome if ultrasound examination does not show any fetal abnormality, provided that the center has accumulated enough experience with the technique of amnio-PCR and is confident of the standard of ultrasound examination. The risk of missing clinically significant chromosomal abnormalities is very small. Furthermore, this approach might be more effective in terms of anxiety relief for women with false-positive Down syndrome screening.


   Footnotes
 
doi:10.1016/S0029-7844(03)00222-9

Received June 27, 2002. Received in revised form September 26, 2002. Accepted November 21, 2002.


   REFERENCES
TOP
ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Cuckle H. Biochemical screening for Down syndrome. Eur J Obstet Gynecol Reprod Biol 2000;92:97–101.[Medline]

2. Von Eggeling F, Freytag M, Fahsold R, Horsthemke B, Claussen U. Rapid detection of trisomy 21 by quantitative PCR. Hum Genet 1993;91:567–70.[Medline]

3. Pertl B, Yau SC, Sherlock J, Davies AF, Mathew CG, Adinolfi M. Rapid molecular method for prenatal detection of Down’s syndrome. Lancet 1994;343:1197–8.[Medline]

4. Verma L, Macdonald F, Leedham P, McConachie M, Dhanjal S, Hulten M. Rapid and simple prenatal DNA diagnosis of Down’s syndrome. Lancet 1998;352:9–12.[Medline]

5. Leung WC, Lam YH, Wong Y, Lau ET, Tang MHY. The effect of fast reporting by amnio-PCR on anxiety levels in women with positive biochemical screening for Down syndrome–a randomized controlled trial. Prenat Diagn 2002;22:256–9.[Medline]

6. Pertl B, Pieber D, Lercher-Hartlieb A, Orescovic I, Haeusler M, Winter R, et al. Rapid prenatal diagnosis of aneuploidy by quantitative fluorescent PCR on fetal samples from mothers at high risk for chromosome disorders. Mol Hum Reprod 1999;5:1176–9.[Abstract/Free Full Text]

7. Levett LJ, Liddle S, Meredith R. A large-scale evaluation of amnio-PCR for the rapid prenatal diagnosis of fetal trisomy. Ultrasound Obstet Gynecol 2001;17:115–8.[Medline]

8. Mann K, Fox SP, Abbs SJ, Yau SC, Scriven PN, Docherty Z, et al. Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis. Lancet 2001;358:1057–61.[Medline]

9. Adinolfi M, Sherlock J. Prenatal detection of chromosome disorders by QF-PCR. Lancet 2001;358:1030–1.[Medline]

10. Marteau TM. Prenatal testing: Towards realistic expectations of patients, providers and policy makers. Ultrasound Obstet Gynecol 2002;19:5–6.[Medline]

11. Warburton D. De novo balanced chromosome rearrangements and extra marker chromosomes identified at prenatal diagnosis: Clinical significance and distribution of breakpoints. Am J Hum Genet 1991;49:995–1013.[Medline]




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