Significance of a False-Positive Trisomy 18 Multiple-Marker Screening Test

Significance of a False-Positive Trisomy 18 Multiple-Marker Screening Test KATHARINE D. WENSTROM, MD, JOHN OWEN, MD, CYNTHIA G. BRUMFIELD, MD, RICHARD O. DAVIS, MD, MARY DUBARD, MA, AND TED GARCIA, MD Objective: To determine if a false-positive trisomy 18 multiple-marker screening test (all three analytes low: maternal serum alpha-fetoprotein [AFP] at most 0.75 multiples of the median [MoM], unconjugated estriol at most 0.60 MoM, and hCG at most 0.55 MoM) indicates increased risk for obstetric complications or is related to maternal weight. Methods: We accessed our genetic database to obtain multiple-marker screening test results, fetal karyotypes, and pregnancy outcomes from all patients with a normal multiple-marker screening test (n 5 3900) and from all patients with a positive trisomy 18 screening test (n 5 103) seen in the prenatal diagnosis clinic from 1992 to 1996. During this period, only maternal serum AFP was adjusted for maternal weight. Results: A positive trisomy 18 screen identified five of 12 trisomy 18 fetuses. Women with a false-positive trisomy 18 screen were heavier (175.6 6 43.8 lb versus 159.9 6 37.9 lb, P < .001) and younger (29.7 6 6.5 years versus 32.3 6 6.5 years, P < .001) than women with a normal multiple-marker screening test, but were not at increased risk for pregnancy complications. Weight-adjusting all three analytes reduced the false-positive trisomy 18 screen rate by 42% (from 1.9% to 1.1%) but did not change the trisomy 18 detection rate. Conclusion: A false-positive trisomy 18 screening test does not indicate increased risk to develop pregnancy complications and may be related to inadequate correction for increased maternal weight. (Obstet Gynecol 1997;90:938 – 42. © 1997 by The American College of Obstetricians and Gynecologists.)

The multiple-marker screening test for Down syndrome (maternal serum alpha-fetoprotein [AFP], unconjugated estriol, hCG, and maternal age) identifies women at risk of having a fetus with either Down syndrome or trisomy 18 (Canick JA, Palomaki GE, Osathanondh R. Prenatal screening for trisomy 18 in the second trimesFrom the Center for Obstetric Research, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, The University of Alabama at Birmingham, Birmingham, Alabama. Supported in part by the Agency for Health Care Policy and Research contract no. DHHS 290-92-0055.

938 0029-7844/97/$17.00 PII S0029-7844(97)00478-X

ter [letter]. Prenat Diagn 1990;10:546 – 8).1 These disparate conditions are associated with two distinct patterns of maternal serum analyte changes: fetal Down syndrome results in lower AFP and estriol and higher hCG levels than expected for gestational age, whereas trisomy 18 is associated with low levels of all three analytes. In the Down syndrome pattern, all three analytes are present at levels generally associated with a pregnancy 2 weeks less mature than the true gestational age. In fact, overestimation of gestational age is a common reason to have a false-positive initial test result.2 However, incorrect dating cannot account for the pattern of analyte changes associated with trisomy 18 pregnancies. There is no time in a normal pregnancy at which all three analyte levels are low (Figure 1).3 Less than 2% of all second-trimester multiple-marker screening tests indicate increased risk for trisomy 18, and only 4 – 6% of screen-positive women actually have an affected fetus. The significance of a false-positive trisomy 18 screening test is unclear. Unusually low levels of placentally derived analytes such as hCG or estriol might indicate placental dysfunction and an increased risk for certain pregnancy complications. On the other hand, the problem may lie not with the pregnancy but with some aspect of the serum analysis or interpretation. Efforts to learn more about the etiology and prognosis of a false-positive trisomy 18 screening test have been hampered by the low incidence of both this test result and trisomy 18 itself (1 in 8000 live births).4 We used our genetic database to determine the significance of a false-positive trisomy 18 screening test and to identify factors that might contribute to this result.

Materials and Methods With Institutional Review Board approval, we accessed our genetic research database to obtain multiple-marker screening test results, fetal karyotypes, and pregnancy

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Figure 1. Median maternal serum concentration by gestational age. AFP 5 alpha-fetoprotein; uE3 5 unconjugated estriol. Published with permission from James DK, Steer PJ, Weiner CP, Gonik B. High risk pregnancy: Management options. Philadelphia: WB Saunders Co., 1994.

outcome data from all women seen in our prenatal diagnosis clinic from 1992 to 1996. Consenting women agreed to have blood drawn for research purposes before scheduled amniocenteses. Seventy-five percent of patients were referred for advanced maternal age, 16% for abnormal maternal serum screening, and 9% for other reasons. The women undergoing evaluation in this clinic were mainly middle class and had health insurance, 90% were white, and nearly all returned to their private obstetricians for management of the remainder of their pregnancy. Pregnancy outcome was assessed by visiting the referring doctors’ offices to review medical records, by calling referring physicians to obtain follow-up, and in the case of a delivery at our hospital, by reviewing our own computerized pregnancy outcome database. All serum was obtained at 14 –20 weeks’ gestation

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and analyzed using commercially available radioimmunoassay kits (maternal serum AFP by Sanofi Pasteur, Chaska, MN; hCG by Nichols Institute, San Juan Capistrano, CA; unconjugated estriol by Diagnostic Systems Laboratory, Webster, TX). Maternal serum AFP levels were corrected routinely for maternal weight and the presence of insulin-dependent diabetes; AFP and hCG were corrected for maternal race. Analyte levels were expressed as multiples of the median (MoM), determined by comparison with the normal medians established in the patient population served by the University of Alabama. The weight correction formula was derived by fitting a regression equation to the distribution of AFP MoMs in this population. The AFP MoM expected for each patient’s weight was determined using the formula. The actual MoM was then divided by the expected MoM to yield the weightcorrected MoM.5 The same method was used ultimately to establish weight correction formulas for estriol and hCG. The test was considered trisomy 18 –screen-positive if all three analytes were at or below the following values: AFP at most 0.75 MoM, unconjugated estriol at most 0.60 MoM, and hCG at most 0.55 MoM (Canick JA, Palomaki GE, Osathanondh R. Prenatal screening for trisomy 18 in the second trimester [letter]. Prenat Diagn 1990;10:546 – 8). The test was considered Down syndrome–screen-positive if the final Down syndrome risk (maternal age–related risk modified by the analytedetermined derived likelihood ratio) was at or above 1:190. This Down syndrome risk cutoff was chosen because it combined an acceptable detection rate (at or above 60%) with a low screen-positive rate (at or above 5%). All women with AFP levels of at least 2.5 MoM were considered neural tube defect–screen-positive. We selected two populations for study: all women with positive trisomy 18 screening tests, and all with test results indicating no increased risk for Down syndrome, trisomy 18, or a neural tube defect. Patient characteristics were considered and pregnancy outcomes compared. Statistical analysis was done using Wilcoxon rank sum test, Student t test, linear regression, Pearson correlation analysis, and x2 when appropriate; P # .05 was considered significant.

Results A total of 5395 patients were entered in the database during the period studied; 68 patients were missing crucial data (maternal weight or analyte levels), leaving 5327 for analysis. Three thousand nine hundred (72%) had test results indicating no increased risk, 103 (2%) had results indicating increased risk for trisomy 18, 1015 (19%) were Down syndrome–screen-positive, and

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False-Positive Trisomy 18 Screen

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Table 1. Patient Characteristics

Table 2. Effect of Weight Correction on Trisomy 18 Screening Test

Negative screen (n 5 3900)

False-positive trisomy 18 screen (n 5 98)

P

32.3 6 6.5 159.9 6 37.9

29.7 6 6.5 175.6 6 43.8

, .001 , .001

23 77

31 69

.04*

24 76

31 69

.12

Maternal age (y) Maternal weight (lb) Race (%) Black Nonblack Payor status (%) Public Private

Weight correction

Data are expressed as mean 6 standard deviation or percent. * This difference was eliminated by weight correction.

309 (6%) were neural tube defect–screen-positive. The mean (6 standard deviation) maternal age of the population was 32.5 6 6.7 years, accounting for the relatively high Down syndrome screen-positive rate. The entire study population included 12 fetuses with trisomy 18. The relatively high incidence of positive trisomy 18 screening tests and trisomy 18 fetuses likely reflects the older maternal age and the selected nature of the study population. Five of the 12 trisomy 18 cases would have been identified by a positive trisomy 18 screen; the remainder had normal test results. One case of trisomy 18 was detected for every 21 amniocenteses indicated by a positive screening test (five of 103). We compared the maternal characteristics of the 98 women who had false-positive trisomy 18 screening tests with those of the 3900 who had results indicating no increased risk (Table 1). Women in the two groups were similar with respect to payor status, but women with false-positive trisomy 18 screens were significantly heavier and younger. A significantly greater proportion of women with false-positive trisomy 18 screens were black (31%) compared with the proportion of women with negative screens who were black (23%, P 5 .04). Because of the association between a false-positive trisomy 18 screening test and increased maternal weight, we investigated the potential effect of weight adjustment of all three analytes (as opposed to weight adjustment of AFP only, the standard during the years of data collection). A weight correction formula was derived for both hCG and unconjugated estriol by fitting an equation to each of the distributions of estriol and hCG MoMs in our entire database (all multiplemarker screening tests performed at our institution for research purposes without regard to the availability of pregnancy follow-up, n 5 8249). These formulas were then applied to the study database, and their effect on the trisomy 18 –screen-positive rate was determined (Table 2). Weight-adjusting unconjugated estriol and hCG in addition to AFP resulted in the normalization of

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Screen positive (n) Screen positive rate (%) Detection rate Cases detected/amnio

AFP only

All analytes

103 1.9 42% (5/12) 1/21

61 1.1 42% (5/12) 1/12

AFP 5 alpha-fetoprotein; Amnio 5 amniocenteses performed.

43 positive trisomy 18 screens and changed one previously negative screen to positive (net change: 103 2 42 5 61 positive screens). This reduced the trisomy 18 –screen-positive rate by 42% (from 1.9% to 1.1%) and reduced the number of amniocenteses required per case detected (from one in 21 to one in 12) but did not change the trisomy 18 detection rate. Weight-adjusting all three analytes also eliminated the racial difference observed in the initial analysis of maternal characteristics (Table 1). Pregnancy outcomes were then assessed (Table 3). Women with negative screening tests (n 5 3900) were compared with all women with false-positive trisomy 18 tests (n 5 98) and with women who remained trisomy 18 –screen-positive after weight correction of all three analytes (n 5 61). Four percent of false-positive trisomy 18 screening tests were due to unsuspected nonviable pregnancies at the time of maternal serum screening (fetal death confirmed at initial ultrasound evaluation). Women with documented viable pregnancies and false-positive trisomy 18 screens (either before or after weight correction) were at no increased risk to have a stillbirth (spontaneous pregnancy loss after 20 weeks’ gestation or neonatal demise), preterm birth (before 37 weeks’ gestation), premature rupture of the membranes, preeclampsia, or intrauterine growth restriction (birth weight below the 10th percentile for Table 3. Incidence of Pregnancy Complications False-positive trisomy 18 (weight Negative False-positive corrected) trisomy 18 screen (n 5 61) (n 5 3900) (all) (n 5 98) Mean maternal weight (lb) Complications (%) Fetal death (at time of serum sample) Fetal growth restriction Stillbirth Preterm delivery PROM Preeclampsia

160 0.9 3.3 1.1 16.6 1.9 1.2

176 4.0* 4.5 1.0 15.0 1.0 3.0

160 4.3* 4.9 1.5 15.7 0 2.9

PROM 5 premature rupture of the membranes. * Significant difference from negative screen.

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gestational age6) when compared with women with screening tests indicating no increased risk.

Discussion Maternal serum AFP screening for fetal neural tube defects was adopted nationwide in the early 1980s.7,8 Relatively early in the experience with this screening test, it became clear that women with false-positive test results (elevated AFP not associated with a fetal defect) were at increased risk to have pregnancy complications such as intrauterine growth restriction, preterm delivery, and fetal death.9 –11 Alpha-fetoprotein is produced in the fetal liver and then passes through the placenta to the maternal circulation. It is presumed that women with unexplained elevated AFP levels have some placental damage or a breech in the maternal-placental barrier that results in transplacental passage of a greater amount of AFP than usual and are predisposed to pregnancy complications associated with placental dysfunction.12 Similarly, evidence now suggests that unexplained elevated hCG also predicts poor pregnancy outcome, including preterm delivery, lower birth weight, and preeclampsia.13,14 Human chorionic gonadotrophin is produced by placental cytotrophoblast. In vitro data support the hypothesis that early placental vascular damage leading to decreased oxygen supply results in increased hCG production by hyperplastic cytotrophoblast cells.15 Thus, rather than reflecting placental damage, it is likely that hCG levels rise in response to such damage. When both AFP and hCG levels are elevated, the risk for a poor pregnancy outcome is especially high.16 Less is known about unexpectedly low analyte levels. In the past, low third-trimester levels of estriol and hCG were thought to predict placental dysfunction and impending fetal distress,17,18 but similar associations have not been demonstrated in the second trimester. However, because there is no time in a normal pregnancy when AFP, unconjugated estriol, and hCG levels are all low, this relatively infrequent screening test result (a positive trisomy 18 screen) raises questions about fetal and placental health. Our results indicate that 4% of positive trisomy 18 screening tests are due to nonviable pregnancies at the time of serum sampling. It is likely that these women had serum drawn for screening purposes before a normal ongoing pregnancy could be confirmed. Forty percent of positive tests were related to inadequate correction for maternal weight. Large women have large volumes of distribution. Analytes produced at a fairly constant rate by the fetoplacental unit are diluted in the expanded maternal circulation, with resulting

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low maternal serum levels. Because the mean maternal weight of the women who remained screen-positive after weight correction was the same as the weight of the women at no increased risk, it is likely that our weight correction formulas were adequate. However, maternal weight and nonviable pregnancy cannot be the only causative factors. One percent of trisomy 18 screening tests in our database were still positive after weight correction. Because we found no association between false-positive test results and pregnancy complications, it seems unlikely that fetoplacental pathology plays a factor. It is possible that some women of normal weight with false-positive trisomy 18 tests have a larger than expected volume of distribution. Unfortunately, we were unable to evaluate other serum analytes (eg, urea nitrogen or creatinine), which might have helped us assess this possibility. It is also possible that in some women, the maternal-placental interface is such that fetoplacental products produced in normal quantities pass less readily into the maternal circulation. Our data indicate that confirmation of fetal viability and weight correction of all three maternal serum analytes will reduce substantially the number of falsepositive trisomy 18 screening tests. After amniocentesis has been performed and a normal fetal karyotype has returned, women with false-positive trisomy 18 screening tests can be reassured that they are not at increased risk of having pregnancy complications.

References 1. American College of Obstetricians and Gynecologists. Down syndrome screening. ACOG Committee opinion no. 141. Washington, DC: American College of Obstetricians and Gynecologists, 1994. 2. Haddow JE, Palomaki GE, Knight GJ, Williams J, Pulkkinen A, Canick JA, et al. Prenatal screening for Down’s syndrome with use of maternal serum markers. N Engl J Med 1992;327:588 –93. 3. Williamson RA. Abnormalities of alpha-fetoprotein and other biochemical tests. In: James DK, Steer PJ, Weiner CP, Gonik B, eds. High risk pregnancy management options. London: WB Saunders, 1995:651. 4. Hook EB, Hammerton JL. The frequency of chromosome abnormalities detected in consecutive newborn studies— differences between studies—results by sex and by severity of phenotypic involvement. In: Hook EB, Porters IH, eds. Population cytogenetic studies in humans. New York: Academic Press, 1977:63. 5. Knight GJ, Palomaki GE, Hddow JE. Use of maternal serum alpha-fetoprotein measurements to screen for Down’s syndrome. Clin Obstet Gynecol 1988;31:206 –27. 6. Brenner WE, Edelman DA, Hendricks CH. A standard of fetal growth for the United States of America. Am J Obstet Gynecol 1976;126:555– 64. 7. Haddow JE, Kloza EM, Smith DW, Knight GJ. Data from an alpha-fetoprotein pilot screening program in Maine. Obstet Gynecol 1983;62:556 – 60. 8. Burton BK, Sowers SG, Nelson LH. Maternal serum a-fetoprotein

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screening in North Carolina: Experience with more than twelve thousand pregnancies. Am J Obstet Gynecol 1983;146:439 – 44. Brock DJH, Barron L, Jelen P, Watt M, Scrimgeour JB. Maternal serum alpha-fetoprotein measurements as an early indicator of low birth-weight. Lancet 1977;ii:267– 8. Robinson L, Grau P, Crandall BF. Pregnancy outcomes after increasing maternal serum alpha-fetoprotein levels. Obstet Gynecol 1989;74:17–20. Silver RM, Draper ML, Byrne JL, Ashwood EA, Lyon JL, Branch DW. Unexplained elevations of maternal serum alpha-fetoprotein in women with antiphospholipid antibodies: A harbinger of fetal death. Obstet Gynecol 1994;83:150 –5. Berkeley AS, Killackey MA, Cederqvist LL. Elevated maternal serum a-fetoprotein levels associated with breakdown in fetalmaternal-placental barrier. Am J Obstet Gynecol 1983;146:859 – 61. Sorensen TK, Williams MA, Zingheim RW, Clement SJ, Hickok DE. Elevated second-trimester human chorionic gonadotropin and subsequent pregnancy-induced hypertension. Am J Obstet Gynecol 1993;169:834 – 8. Wenstrom KD, Owen J, Boots LR, DuBard MA. Elevated secondtrimester human chorionic gonadotropin levels in association with poor pregnancy outcome. Am J Obstet Gynecol 1994;171:1038 – 41. Fox H, Path MC. Effect of hypoxia on trophoblast in organ culture. A morphologic and autoradiographic study. Am J Obstet Gynecol 1970;7:1058 – 64. Benn PA, Horne D, Briganti S, Rodis JF, Clive JM. Elevated

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second-trimester maternal serum hCG alone or in combination with elevated alpha-fetoprotein. Obstet Gynecol 1996;87:217–22. 17. Kundu N, Carmody PJ, Didolkar SM, Petersen LP. Sequential determination of serum human placental lactogen, estriol, and estetrol for assessment of fetal morbidity. Obstet Gynecol 1978;52: 513–20. 18. Obiekwe BC, Chard T. Human chorionic gonadotropin levels in maternal blood in late pregnancy: Relation to birthweight, sex and condition of the infant at birth. Br J Obstet Gynaecol 1982;89:543– 6.

Address reprint requests to:

Katharine D. Wenstrom, MD Department of Obstetrics and Gynecology The University of Alabama at Birmingham 618 South 20th Street, UAB Station Birmingham, AL 35233-7333

Received May 14, 1997. Received in revised form July 17, 1997. Accepted August 7, 1997. Copyright © 1997 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.

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