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ORIGINAL RESEARCH |
From the Division of Human Genetics, Department of Pediatrics, University of Connecticut Health Center, Farmington; and Division of Maternal–Fetal Medicine, Department of Obstetrics and Gynecology, St. Francis Hospital and Medical Center, Hartford, Connecticut.
Address reprint requests to: Peter A. Benn, PhD, Division of Human Genetics, Department of Pediatrics, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-6140; E-mail: benn{at}nso1.uchc.edu.
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ABSTRACT |
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 ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: All quadruple tests performed on singleton pregnancies over a 32-month period were reviewed. The sensitivity and false-positive rates were compared with the theoretic rates predicted by the screening model when applied to a population of women with the same maternal age distribution.
RESULTS: Twenty-three thousand seven hundred four women with unaffected pregnancies and 45 women with Down syndrome–affected pregnancies received the quadruple test. Mean analyte values for both unaffected and affected pregnancies were similar to those expected. The sensitivity of the quadruple test, which was based on ascertainment of all viable affected pregnancies in the screened population, was 85.8%. This sensitivity did not significantly differ from an expected 83.8% (P = .8). The initial false-positive rate, 9.0%, was significantly below that expected (9.9%) (P = .002) and was further reduced to 8.2% after correction for major gestational age errors. The positive predictive value of the quadruple test was one in 51. Women with an affected pregnancy and a positive test result (true positives) generally had very high risks (median 1:22) relative to women with false-positive results (median risk 1:111).
CONCLUSION: The quadruple test meets or exceeds performance expectations and appears to represent an improvement over the widely used triple test.
Women carrying Down syndrome–affected fetuses often show specific patterns for maternal serum analyte concentrations that differ from those seen in unaffected pregnancies.1 These differences can be combined with maternal age-specific risks to provide screening protocols for Down syndrome. In the second trimester, measurement of maternal serum alpha-fetoprotein (AFP), unconjugated estriol (E3), and human chorionic gonadotropin (hCG) plus maternal age is used in a widely accepted protocol for the identification of pregnancies at high risk for fetal Down syndrome.2–4 This triple test has a 71.3% sensitivity at a 5% false-positive rate for pregnancies in which the gestational age is established by ultrasound.5 In practice, the sensitivity and false-positive rate are determined by the risk cutoff chosen for screening and the maternal age distribution of the screened population. For example, using a second-trimester risk cutoff of 1:270, 77.9% of Down syndrome–affected pregnancies can be detected with a 7.8% false-positive rate when the triple test is applied to the 1999 US pregnancy population.5 The validity of the triple test has been demonstrated in a large number of retrospective and prospective studies.6
The potential use of inhibin in second-trimester screening for Down syndrome was first suggested by Van Lith et al.7 By computer modeling, the combination of maternal serum AFP, E3, hCG, and the ßA subunit of inhibin, termed inhibin-A, can be shown to improve the sensitivity and lower the false-positive rate of the screening.8,9 The expected sensitivity for this quadruple test is 77.5% when the false-positive rate is held at 5%.5 Applied to the 1999 US population, this should achieve an 81.5% detection rate with a 6.9% false-positive rate. However, thus far, there are no detailed reports of large trials documenting the performance of the quadruple test. Wald and Huttly10 noted that quadruple test risks calculated in 19,397 pregnancies were consistent with expectations, but their Down syndrome screening program’s sensitivity and false-positive rate were not reported.
We report our experience of screening over 23,000 women using the second-trimester quadruple test.
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MATERIALS AND METHODS |
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ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A second-trimester Down syndrome risk greater or equal to 1:270 was used to define a screen-positive result. Maternal age-specific prevalence rates for Down syndrome at birth13 were adjusted to the second trimester using a factor of 0.76 for the survival of an affected fetus14 and 0.985 for an unaffected fetus.15 During the study period, revised statistical parameters were introduced into the screening program.16 However, for the purposes of this evaluation, all risks were calculated using a common set of means, standard deviations, correlation coefficients, and truncation limits for the analytes.8,16,17 Recalculated risks using the revised statistical parameters were not used for patient counseling.
Expected maternal age-specific sensitivity and false-positive rates for the quadruple test and triple test were established by computer simulations using a program developed for S-plus (MathSoft Inc., Seattle, WA).18 These rates were separately established for pregnancies dated by ultrasound and by time from the last menstrual period (LMP). Overall sensitivity and false-positive rates for the screened population were then determined by calculating averages, weighed for the number of women screened at each age and the method of pregnancy dating.
Affected pregnancies were identified through a review of cytogenetic laboratory records, regional genetic consultation records, ultrasound reports, and follow-up gathered from referring physician offices. We have previously shown that this can result in a comprehensive ascertainment of affected pregnancies.19 The collection of the follow-up data was approved by the University of Connecticut Investigational Review Board.
Statistical tests were performed using SPSS (SPSS Inc., Chicago, IL). For tests of significance a P value of less than .05 was considered to be significant.
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RESULTS |
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ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Within the screened population, a total of 45 cases of Down syndrome were identified. In 33 cases (30 screen positive and three screen negative), the diagnosis of trisomy 21 was established from cytogenetic analysis of amniotic fluid cells. In 12 cases (nine screen positive and three screen negative), diagnosis was established at birth and confirmed by cytogenetic analysis of blood. An adjustment was made for cases of Down syndrome that result in fetal death without being recognized.20 Based on the estimate that only 76% of the Down syndrome–affected pregnancies would survive to term, the 12 cases identified at birth would be equivalent to 12/0.76 = 16 present in the second trimester. Combined with the 33 diagnosed through amniocentesis, the adjusted number of affected pregnancies was therefore 33 + 16 = 49. Assuming random case referral for screening, and based on the number of women screened for each maternal age, the theoretic number of Down syndrome–affected pregnancies that should have been present in the 23,749 test referrals was 54. The difference between the 49 affected pregnancy cases accounted for in this audit and the theoretic 54 cases expected was not statistically significant (Pearson 
21 0.62, P = .62).
Table 1
summarizes the overall results of the screening. Mean maternal serum analyte values, expressed as multiples of the median (MoM), for the 45 Down syndrome–affected pregnancies were close to published means derived from unselected affected pregnancies (maternal serum AFP 0.72 MoM, E3 0.72 MoM, hCG 2.01 MoM, inhibin-A 1.79 MoM).17 Without adjustment for fetal losses from the time of screening to full term, the overall sensitivity of the quadruple test screening was 39 of 45 or 86.7% (95% confidence interval [CI] 73.2%, 95%). With this adjustment in both screen-positive and screen-negative women, the sensitivity was 85.8% (95% CI 72.8%, 94.0%), which did not significantly differ from the expectation of 83.8% derived from the computer modeling (Pearson 21 0.08, P = .8). Using the theoretic number of 54 affected pregnancies in the screened population, the sensitivity estimate was 77.7% (95% CI 64.4%, 88.0%). The median quadruple test risks were 1:30 for all Down syndrome–affected pregnancies and 1:22 for the subgroup with positive screening results. The amniocentesis rate in women with an affected pregnancy and a screen-positive test result was 30 of 39 or 77% (95% CI 61%, 89%).
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21 9.8, P = .002). The false-positive rate after correction for major gestational age errors was 8.2% (95% CI 7.9%, 8.6%). The median quadruple test risks were 1:5992 for all unaffected pregnancies and 1:111 for false-positives. The amniocentesis rate for the false-positives was 663 of 1946 or 34% (95% CI 32%, 36%).
Table 2 summarizes the expected performance of screening using maternal age, the triple test, and the quadruple test. These rates apply to a population of women with the same maternal age distribution as that which actually received the quadruple test.
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DISCUSSION |
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Validation of the sensitivity for any prenatal screening protocol is problematic because the number of affected pregnancies in the study population is usually small and there is uncertainty as to the precise prevalence of Down syndrome fetuses at various times in pregnancy.21 Additionally, the availability of alternative screening and testing options can distort any estimates of the prevalence in the screened population. For example, the increased use of sonography to identify fetal structural anomalies or markers compatible with Down syndrome may result in some high-risk women opting for amniocentesis without receiving second-trimester serum screening. The number of affected pregnancies expected to be present in the screened population is therefore uncertain and cannot be used to reliably calculate sensitivity. Furthermore, it is rarely possible to be completely certain that all Down syndrome pregnancies have been ascertained through follow-up data collection. Thus, our estimate of the sensitivity of the screening needs to be interpreted cautiously. The fact that observed median MoM values for the individual analytes are close to those expected provides some additional reassurance that there was no systematic under-ascertainment of screen-negative affected pregnancies.
The false-positive rate is not subject to the same problems with ascertainment or prevalence and is based on a large sample size. The false-positive rate was somewhat lower than that predicted by computer modeling. This presumably reflects minor differences in the standard deviations and correlation coefficients for the analytes, relative to published values for these variables. This, in turn, may reflect differences in the accuracy of gestational age assessments, population characteristics, and assay conditions.
The quadruple test shows a higher sensitivity and lower false-positive rate than the triple test (Table 2
) and appears to be economically justifiable on the basis of this improved performance.8,9 Relative to screening with the triple test, the use of the quadruple test should result in greater separation of the affected and unaffected pregnancy risk distributions. Generally, higher risks are reported for affected pregnancies and lower risks for unaffected pregnancies. Within the group of women with screen-positive test results, higher risks are associated with increased amniocentesis utilization rates.22 An additional indirect benefit with the quadruple test therefore lies in more effective use of amniocentesis. We conclude that inhibin-A should be widely available for second-trimester Down syndrome screening.
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Footnotes |
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Received November 18, 2002. Received in revised form December 18, 2002. Accepted December 26, 2002.
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REFERENCES |
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2. Wald NJ, Cuckle HS, Densem JW, Nanchahal K, Royston P, Chard T, et al. Maternal serum screening for Down’s syndrome in early pregnancy. BMJ 1988;297:883–7.
3. Palomaki GE, Knight GJ, McCarthy J, Haddow JE, Donhowe JM. Maternal serum screening for Down syndrome in the United States: A 1995 survey. Am J Obstet Gynecol 1997;176:1046–51.[Medline]
4. Egan JFX, Kaminsky LM, DeRoche ME, Barsoom MJ, Borgida AF, Benn PA. Antenatal Down syndrome screening in the U. S. in 2001: A survey of maternal-fetal medicine specialists. Am J Obstet Gynecol 2002;187:1230–4.[Medline]
5. Benn PA, Lillian M, Kaminsky LM, Ying J, Borgida AF, Egan JFX. Combined second trimester biochemical and ultrasound screening for Down syndrome. Obstet Gynecol 2002;100:1168–76.
6. Cuckle H. Established markers in second trimester maternal serum. Early Hum Dev 1996;47:S27–9.
7. Van Lith JM, Pratt JJ, Beekhuis JR, Mantingh A. Second-trimester maternal serum immunoreactive inhibin as a marker for fetal Down’s syndrome. Prenat Diagn 1992;12: 801–6.[Medline]
8. Wald NJ, Densem JW, George L, Muttukrishna S, Knight PG. Prenatal screening for Down’s syndrome using inhibin-A as a serum marker. Prenat Diagn 1996;16:143–52. Erratum: Prenat Diagn 1997;17:285–90.[Medline]
9. Haddow JE, Palomaki GE, Knight GJ, Foster DL, Neveux LM. Second trimester screening for Down syndrome using maternal serum inhibin A. J Med Screen 1998;5:115–9.
10. Wald NJ, Huttly WJ. Validation of risk estimation using the quadruple test in prenatal screening for Down syndrome. Prenat Diagn 1999;19:1081–2.[Medline]
11. Benn PA, Horne D, Briganti S, Greenstein RM. Prenatal diagnosis of diverse chromosome abnormalities in a population of patients identified by triple marker testing as screen-positive for Down’s syndrome. Am J Obstet Gynecol 1995;173:496–501.[Medline]
12. Benn PA, Borgida A, Horne D, Briganti S, Collins R, Rodis JF. Down syndrome and neural tube defect screening: The value of using gestational age by ultrasonography. Am J Obstet Gynecol 1997;176:1056–61.[Medline]
13. Cuckle HS, Wald NJ, Thompson SG. Estimating a woman’s risk of having a pregnancy associated with Down’s syndrome using her age and serum alpha-fetoprotein level. Br J Obstet Gynaecol 1987;94:387–402.[Medline]
14. Benn PA, Egan JFX. Survival of Down syndrome in utero. Prenat Diagn 2000;20:432–9.
15. Egan JFX, Benn P, Borgida AF, Rodis JF, Campbell WA, Vintzileos AM. Efficacy of screening for fetal Down syndrome in the United States from 1974 to 1997. Obstet Gynecol 2000;96:979–85.
16. Wald NJ, Hackshaw AK, George LM. Assay precision of serum 
fetoprotein in antenatal screening for neural tube defects and Down’s syndrome. J Med Screen 2000;7:74–7.
17. Wald NJ, Densem JW, Smith D, Klee GG. Four-marker serum screening for Down’s syndrome. Prenat Diagn 1994;14:707–16.[Medline]
18. Benn PA, Ying J, Beazoglou T, Egan JFX. Estimates for the sensitivity and false-positive rates for second trimester serum screening for Down syndrome and trisomy 18 with adjustment for cross-identification and double-positive results. Prenat Diagn 2001;21:46–51.[Medline]
19. Benn PA. Preliminary evidence for associations between second-trimester human chorionic gonadotropin and unconjugated oestriol levels with pregnancy outcome in Down syndrome pregnancies. Prenat Diagn 1998;18: 319–24.[Medline]
20. Palomaki G, Neveux LM, Haddow JE. Can reliable Down’s syndrome detection rates be determined from prenatal screening and intervention trials? J Med Screen 1996;3:12–7.[Medline]
21. Cuckle H. Down syndrome fetal loss rate in early pregnancy. Prenat Diagn 1999;19:1177–9.
22. Chen J, Heffley D, Beazoglou T, Benn PA. Utilization of amniocentesis by women screening positive for Down syndrome on the second trimester triple test. Commun Genet 2000;3:24–30.
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P. A. Benn, J. F. X. Egan, M. Fang, and R. Smith-Bindman Changes in the Utilization of Prenatal Diagnosis Obstet. Gynecol., June 1, 2004; 103(6): 1255 - 1260. [Abstract] [Full Text] [PDF]
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