Estimates for the sensitivity and false-positive rates for secondtrimester serum screening for Down syndrome and trisomy 18with adjustment for cross-identification and double-positiveresults

Peter A. Benn1*, Jun Ying2, Tryfon Beazoglou3 and James F. X. Egan4

1Division of Human Genetics, Department of Pediatrics, University of Connecticut Health Center, Farmington, CT 06030,USA2Department of Statistics, University of Connecticut, Storrs, CT 06269, USA3Department of Pediatric Dentistry, University of Connecticut Health Center, Farmington, CT 06030, USA4Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, University of Connecticut Health Center,Farmington, CT 06030, USA

Second trimester screening for fetal Down syndrome and trisomy 18 is available through separate protocolsthat combine the maternal age-specific risk and the analysis of maternal serum markers. We havedetermined the extent to which additional Down syndrome affected pregnancies may be identified throughtrisomy 18 screening, and the extent to which additional cases of trisomy 18 may be screen-positive forDown syndrome. The combined false-positive rate, taking into consideration those pregnancies that arescreen-positive by both protocols, has also been determined. Sensitivity and false-positive rates weredetermined by computer simulation of results that incorporated previously published statistical variablesinto the model. Using second trimester risk cut-offs of 1 : 270 for Down syndrome and 1 : 100 for trisomy 18,it was found that few additional cases of Down syndrome are identified through trisomy 18 screening.However, approximately 610% of trisomy 18 affected pregnancies will be screen-positive for Downsyndrome but screen-negative for trisomy 18. For women aged 40 or more, the false-positive rate fortrisomy 18 exceeds 1% and approximately half of these cases will also be screen-positive for Downsyndrome. For a population with maternal ages equivalent to that in the United States in 1998, afteradjusting for the cross-identification, the sensitivity for three-analyte trisomy 18 screening is 78%. If thistesting is performed in conjunction with Down syndrome triple screening, the Down syndrome sensitivityis 75% and the combined false-positive rate is 8.5%. If the three-analyte trisomy 18 screening is performedwith the Down syndrome quad screen, the trisomy 18 sensitivity remains at 78%, the Down syndromesensitivity is 79%, and combined false-positive rate is 7.5%. Sensitivity and false-positive rates are alsoprovided for other widely used Down syndrome and trisomy 18 risk cut-offs. Sensitivity and false-positiverates that take into consideration cross-identification and double-positives should be helpful for pre-testcounseling and the evaluation of serum screening programs. Copyright # 2001 John Wiley & Sons, Ltd.

KEY WORDS: Down syndrome; trisomy 18; maternal serum screening; second trimester

INTRODUCTION

Trisomy 21 and trisomy 18 are the two most prevalentautosomal trisomies encountered at birth (Hook,1981). Second trimester prenatal screening for bothof these trisomies is available through separateprotocols that combine the maternal age-specific riskwith maternal serum analyte measurements (Waldet al., 1988; Staples et al., 1991; Barkai et al., 1993).Pregnancies complicated by fetal Down syndrome areoften characterized by low levels of maternal serumalpha-fetoprotein (MS-AFP) (Merkatz et al., 1984),low unconjugated estriol (uE3) (Canick et al., 1988),high human chorionic gonadotrophin (hCG) (Bogartet al., 1987) and high inhibin-A (INH-A) (Van Lithet al., 1992). Trisomy 18 pregnancies also show anassociation with low MS-AFP, low uE3 but differ

from Down syndrome pregnancies in that they mayhave a low hCG (Canick et al., 1990).

Previous reports have separately evaluated theefficacy of second trimester three- or four-analytescreening for Down syndrome (Wald et al., 1988;Haddow et al., 1992; Wald et al., 1996; Haddow et al.,1998) and three-analyte screening for trisomy 18(Palomaki et al., 1995; Benn et al., 1999a). BecauseDown syndrome and trisomy 18 affected pregnanciescan share the common characteristics of advancedmaternal age, low MS-AFP and low uE3, there shouldbe some additional affected pregnancies identifiedthrough one or the other screening protocol thatwere not specifically designed for that particularaneuploidy (cross-identification). Furthermore, somepatients will be identified as screen-positive by bothprotocols (double-positives). Amniocentesis shouldbe offered to any woman who is screen-positive byeither protocol (Feuchtbaum et al., 2000) and chromo-some analysis will identify both aneuploidies. Inevaluating the efficacy of screening it is therefore

*Correspondence to: P. Benn, University of Connecticut HealthCenter, Division of Human Genetics, 263 Farmington Avenue,Farmington, CT 06030-6140, USA. E-mail: Benn@nso1.uchc.edu

PRENATAL DIAGNOSIS

Prenat Diagn 2001; 21: 4651.

Copyright # 2001 John Wiley & Sons, Ltd. Received: 14 June 2000Revised: 19 September 2000

Accepted: 22 September 2000

appropriate to consider the overall detection rates ofDown syndrome and trisomy 18 and the effectivefalse-positive rate of the two protocols consideredtogether.

We have carried out a simulation study to estimatethe extent to which additional cases of Downsyndrome and trisomy 18 may be identified throughcross-identification. We also estimate the extent towhich normal pregnancies are identified as double-positives.

MATERIALS AND METHODS

We randomly generated 100 000 sets of analyte valuescorresponding to normal, Down syndrome andtrisomy 18 affected pregnancies using the multivariateGaussian distribution generator in the statisticalcomputer package, S-Plus (Mathsoft, Seattle, WA).The use and validation of this program for Downsyndrome screening has been described by Larsen et al.(1998). For the normal and Down syndrome popula-tion distributions, the means, standard deviations andcorrelation coefficients between the biochemical mar-kers were as determined by Wald et al. (1994, 1996,1997) and for trisomy 18 by Palomaki et al. (1995).INH-A values were not specified for trisomy 18affected pregnancies and were not therefore used inevaluating risk for trisomy 18. Separate simulationswere carried out for pregnancies dated by ultrasoundand those dated on the basis of time since the lastmenstrual period (LMP). Either a triple test combi-nation (MS-AFP, uE3 and hCG) or a quad testcombination (MS-AFP, uE3, hCG and INH-A) wasused for evaluating Down syndrome risk and a three-test protocol (MS-AFP, uE3 and hCG) for trisomy 18.Prior to the computation of the likelihood ratios fromthe randomly generated analyte values, truncationlimits were applied to the analyte values as specifiedfor Down syndrome and trisomy 18 calculations(Wald et al., 1996; Palomaki et al., 1995).

The maternal age-specific prevalence for Downsyndrome was based on the eight-study curve ofBray et al. (i.e. Table III, data excluding Trimble andBaird, Bray et al., 1998). Adjustment to correct forthe prevalence of Down syndrome in the secondtrimester was carried out using a 0.8554 factor thatwas derived by comparing observed rates of fetalDown syndrome at amniocentesis with that predictedfrom the gestational age-adjusted prevalence curve(Cuckle, 1999; Benn and Egan, 2000). The maternalage-specific risk for trisomy 18 was assumed to be one-tenth that for Down syndrome (Hook and Hamerton,1977) and the adjustment factor to establish secondtrimester prevalence (derived by the same method asthat for Down syndrome) was 0.3855.

To determine the sensitivities and false-positiverates, the following method was used. At eachmaternal age, we determined the threshold likelihoodratio needed to generate a second trimester risk greateror equal to 1 : 270 for Down syndrome and also thelikelihood ratio needed for a risk greater or equal to

1 : 100 for trisomy 18. Each combination of analyteresults in the normal, Down syndrome and trisomy 18pregnancy populations was then evaluated to deter-mine which cases had likelihood ratios that exceededthese thresholds (Haddow and Palomaki, 1993). Theproportion of cases with either Down syndromescreening and/or trisomy 18 screening likelihoodratios above these levels defined the sensitivities andfalse-positive rates. Alternative combinations ofsecond trimester risk cut-offs for Down syndrome(1 : 190) and for trisomy 18 (1 : 150 or 1 : 50) were alsoevaluated. We also included full term risk cut-offs of1 : 250 for Down syndrome and 1 : 50 for trisomy 18 asthese represent widely used European standards forpresenting risk. Term risks were converted to secondtrimester risks using the adjustment factors specifiedabove and sensitivities and false-positive rates werethen calculated as described.

Overall performance of the screening tests wereestablished for a population in which 60% ofpregnancies were dated on the basis of ultrasoundmeasurements and 40% by LMP dating (Benn et al.,1997). The 1998 United States final natality statisticswere used as a source for the maternal age distribution(National Center for Health Statistics, 2000).

RESULTS

Table 1 shows the theoretical maternal age-specificsensitivities and false-positive rates for Down syn-drome and trisomy 18 screening achievable with thetriple test. These results pertain to use of a 1 : 270second trimester risk cut-off for Down syndromescreening and a 1 : 100 second trimester risk cut-off fortrisomy 18 screening. The sensitivities and false-positive rates for Down syndrome and trisomy 18screening are provided for the two separate screeningprotocols (columns 25) and also the effective rates arepresented, after cross-detection and double false-positives are taken into consideration (columns 68).The summary rates for women aged less than 35, 35 ormore, and all ages (bottom three rows) are based onthe 1998 United States birth statistics. For thatpopulation, 12.9% of women were aged 35 or more,and 2.1% were aged 40 or more.

Table 2 shows the corresponding sensitivities andfalse-positive rates achieved when the quad test isused. The sensitivity and false-positive rates fortrisomy 18 screening are identical in Tables 1 and 2because INH-A is not used in trisomy 18 screening.

The results presented in Tables 1 and 2 show thatfew additional cases of Down syndrome are identifiedthrough trisomy 18 screening and the effect of thecombined screening does not materially alter theestimates of sensitivity (column 6 vs column 2).However, this is not true for trisomy 18 identifiedthrough Down syndrome screening. An additional610% of trisomy 18 cases will be identified among thegroup of women with positive Down syndromescreening results (column 7 vs column 4). This

ADJUSTED SENSITIVITY AND FALSE-POSITIVE RATES 47

Copyright # 2001 John Wiley & Sons, Ltd. Prenat Diagn 2001; 21: 4651.

effectively raises the trisomy 18 sensitivity to 78% forthe total population screened.

The effect of double identification of false-positivecases only becomes an issue for women at the mostadvanced maternal ages. For women aged 40 or more,the trisomy 18 screen false-positive rate exceeds 1%and increases rapidly with advancing maternal age(column 5). For these women, approximately half willalso be screen-positive for Down syndrome. Theoverall proportion of older women with false-positiveresults (column 8) is therefore less than that expectedfrom the independent evaluations of these screeningtests.

The data presented in Tables 1 and 2 are based on a

population in which 60% of pregnancies were dated byultrasound measurements and 40% by time fromLMP. Table 3 provides summary rates for theperformance of the screening when these two preg-nancy dating methods are considered separately. Thesedata apply to screening based on the 1 : 270 secondtrimester cut-off for Down syndrome 1 : 100 secondtrimester cut-off for trisomy 18. The effect of usingsome other commonly used cut-offs for Downsyndrome and trisomy 18 screening is illustrated inthe summary data provided in Table 4. The maternalage-specific sensitivities and false-positive rates used toderive these summaries of efficacy are available bywriting to the authors.

Table 1Maternal age-specific sensitivities (%) and false-positive rates (FPR) (%) for Down syndrome screening (tripletesting) and trisomy 18 screening with the effective rates after considering cross-identification

Maternal age (at full term)

Separate evaluation of screening Combined evaluation of screening

Down syndrome Trisomy 18 Down syndrome Trisomy 18 Unaffected

Sensitivity FPR Sensitivity FPR Sensitivity Sensitivity FPR

14 50.3 3.38 58.2 0.06 50.3 64.5 3.4315 50.3 3.39 58.2 0.06 50.4 64.5 3.4416 50.4 3.41 58.2 0.06 50.4 64.5 3.4617 50.5 3.43 58.3 0.06 50.5 64.6 3.4818 50.6 3.45 58.3 0.06 50.7 64.7 3.5019 50.8 3.49 58.4 0.06 50.8 64.8 3.5520 51.0 3.55 58.5 0.06 51.1 64.9 3.6021 51.3 3.61 58.6 0.06 51.4 65.0 3.6622 51.7 3.70 58.8 0.06 51.8 65.2 3.7523 52.3 3.81 59.1 0.06 52.3 65.5 3.8624 53.0 3.95 59.3 0.07 53.0 65.8 4.0125 53.8 4.15 59.7 0.07 53.9 66.3 4.2126 54.9 4.43 60.1 0.07 54.9 66.7 4.4927 56.2 4.78 60.8 0.08 56.3 67.4 4.8528 57.9 5.28 61.5 0.09 57.9 68.3 5.3629 59.9 5.89 62.4 0.10 60.0 69.3 5.9730 62.4 6.76 63.4 0.12 62.4 70.4 6.8531 65.1 7.90 64.6 0.13 65.1 71.8 8.0032 68.2 9.37 65.9 0.16 68.2 73.3 9.4833 71.6 11.30 67.2 0.19 71.6 74.9 11.4434 75.1 13.71 68.7 0.24 75.2 76.8 13.8835 78.7 16.66 70.3 0.31 78.8 78.8 16.8736 82.3 20.31 72.0 0.38 82.4 80.8 20.5537 85.6 24.67 73.7 0.51 85.7 82.8 24.9738 88.6 29.72 75.6 0.67 88.7 85.0 30.0839 91.2 35.42 77.6 0.90 91.3 87.1 35.8840 93.4 41.56 79.4 1.20 93.5 89.1 42.1241 95.2 48.09 81.1 1.57 95.2 90.8 48.7842 96.6 54.67 82.6 2.04 96.7 92.4 55.5143 97.7 61.10 83.9 2.71 97.7 93.8 62.1344 98.4 67.23 85.3 3.57 98.5 95.1 68.4245 99.0 72.84 86.6 4.64 99.0 96.2 74.2146 99.3 77.72 87.8 6.01 99.4 97.0 79.2847 99.6 81.99 89.1 7.79 99.6 97.8 83.7448 99.7 85.66 90.3 10.00 99.8 98.4 87.5349 99.8 88.62 91.5 12.69 99.9 98.9 90.62

Total population

DISCUSSION

Our estimates for the maternal age-specific perfor-mance of the triple test in Down syndrome screening(Table 1, columns 2 and 3) appear to be similar toprevious estimates that have been based on a 1 : 270second trimester cut-off (Beazoglou et al., 1998; Bennet al., 1999b). The sensitivities and false-positive ratesalso appear to be consistent with those derived usingalternative second trimester Down syndrome risk cut-offs (Haddow and Palomaki 1993; Reynolds et al.,1993). Minor differences reflect a number of changesin the modeling including the use of updated means,standard deviations, and correlation coefficients. TheDown syndrome birth prevalence curve used by us

also differed from that incorporated in many of theprevious studies and this can alter the estimates ofscreening efficacy (Cuckle, 1998). Our simulation alsoused a different adjustment factor to convert Downsyndrome birth prevalence to second trimester pre-valence, reflecting the need to use a factor that ismatched to the specific birth prevalence curve (Bennand Egan, 2000).

Estimates for the maternal age-specific performanceof the quad test for Down syndrome screening(Table 2, columns 2 and 3) are not available forcomparison. However, the summary estimates forsensitivity and false-positive rates for a totalpopulation are somewhat higher than previouslynoted (Wald et al., 1997; Haddow et al., 1998). This

Table 2Maternal age-specific sensitivities (%) and false-positive rates (FPR) (%) for Down syndrome screening (quadtesting) and trisomy 18 screening with the effective rates after considering cross-identification

Maternal age (at full term)

Separate evaluation of screening Combined evaluation of screening

Down syndrome Trisomy 18 Down syndrome Trisomy 18 Unaffected

Sensitivity FPR Sensitivity FPR Sensitivity Sensitivity FPR

14 59.4 3.30 58.2 0.06 59.4 64.5 3.3515 59.4 3.31 58.2 0.06 59.5 64.5 3.3516 59.5 3.32 58.2 0.06 59.5 64.5 3.3717 59.6 3.34 58.3 0.06 59.6 64.6 3.3918 59.7 3.36 58.3 0.06 59.8 64.7 3.4119 59.9 3.40 58.4 0.06 59.9 64.8 3.4520 60.1 3.45 58.5 0.06 60.1 64.9 3.5021 60.4 3.52 58.6 0.06 60.4 65.0 3.5722 60.7 3.59 58.8 0.06 60.7 65.2 3.6423 61.2 3.69 59.1 0.06 61.2 65.5 3.7524 61.8 3.82 59.3 0.07 61.8 65.8 3.8825 62.6 3.99 59.7 0.07 62.6 66.3 4.0526 63.6 4.23 60.1 0.07 63.6 66.7 4.2927 64.7 4.52 60.8 0.08 64.7 67.4 4.5928 66.1 4.95 61.5 0.09 66.2 68.3 5.0229 67.9 5.48 62.4 0.10 67.9 69.3 5.5630 69.9 6.21 63.4 0.12 69.9 70.4 6.3031 72.1 7.15 64.6 0.13 72.2 71.8 7.2532 74.6 8.39 65.9 0.16 74.7 73.3 8.5133 77.3 9.87 67.2 0.19 77.4 74.9 10.0234 80.1 11.79 68.7 0.24 80.2 76.8 11.9735 82.9 14.07 70.3 0.31 82.9 78.8 14.3036 85.6 16.84 72.0 0.38 85.6 80.8 17.1237 88.1 20.18 73.7 0.51 88.1 82.8 20.5338 90.4 24.01 75.6 0.67 90.5 85.0 24.4539 92.4 28.32 77.6 0.90 92.5 87.1 28.8740 94.1 32.95 79.4 1.20 94.2 89.1 33.6541 95.5 38.08 81.1 1.57 95.6 90.8 38.9342 96.7 43.45 82.6 2.04 96.7 92.4 44.4843 97.6 48.94 83.9 2.71 97.6 93.8 50.2144 98.2 54.58 85.3 3.57 98.3 95.1 56.1245 98.8 60.04 86.6 4.64 98.8 96.2 61.8646 99.1 65.28 87.8 6.01 99.2 97.0 67.4347 99.4 70.04 89.1 7.79 99.5 97.8 72.5148 99.6 74.56 90.3 10.00 99.6 98.4 77.3649 99.7 78.48 91.5 12.69 99.8 98.9 81.59

Total population

can be largely attributed to differences in the maternalage distribution of the population analyzed (Eganet al., 2000) and possibly the other minor differences inthe variables used in the simulation.

Maternal-age specific rates for trisomy 18 screeninghave also not been previously published. Again, theestimates for the performance of trisomy 18 screeningapplied to a total population also appear to becompatible with earlier estimates (Palomaki et al.,1995; Benn et al., 1999a). All projections for theperformance of the screening are based on theassumption that the means, standard deviations,correlation coefficients of analytes, and prevalencecurves are accurate. Because of the rarity of trisomy 18affected pregnancies, these parameters are based onrelatively small numbers. Estimated sensitivity andfalse-positive rates are subject to statistical errorsassociated with the use of finite numbers of cases in the

simulations. These errors will be proportionately moreimportant when low frequency events are considered(e.g. false-positives for trisomy 18 in younger women).For these reasons, the projections for the efficacyof the trisomy 18 screening need to be viewedcautiously.

Our results indicate that separate evaluation ofDown syndrome and trisomy 18 screening results in anunderestimation of the effectiveness in the identifica-tion of trisomy 18 and a very slight overestimation ofthe number of older women with an indication foramniocenteses on the basis of screening alone. Wesuggest that the use of rates that take into considera-tion cross-identification and double-positives are themost appropriate set for pre-test counseling andprogram evaluation because they most accuratelyreflect the overall levels of detection and numbers ofamniocenteses that are indicated at each maternal age.

Table 3Summary sensitivities (%) and false-positive rates (FPR) (%) for Down syndrome and trisomy 18 screening forpregnancies dated by ultrasound (US) and by time from the last menstrual period (LMP)

Test Dating method Maternal age

Separate evaluation of screening Combined evaluation of screening

Down syndrome Trisomy 18 Down syndrome Trisomy 18 Unaffected

Sensitivity FPR Sensitivity FPR Sensitivity Sensitivity FPR

Triple LMP

ACKNOWLEDGEMENTS

Research supported by a grant from the Patrick andCatherine Weldon Donaghue Medical Research Foun-dation.

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Copyright # 2001 John Wiley & Sons, Ltd. Prenat Diagn 2001; 21: 4651.